Conjugation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides

ABSTRACT

The invention provides a process for preparing a conjugate of a  S. aureus  type 5 or type 8 capsular polysaccharide and a carrier molecule, comprising the steps of: (a) depolymerizing the capsular polysaccharide, to give a polysaccharide fragment; (b) oxidizing the fragment in order to introduce an aldehyde group into at least one saccharide residue in the fragment, to give an oxidized saccharide residue; and (c) coupling the oxidized saccharide residue to a carrier molecule via the aldehyde group, thereby giving the conjugate. The coupling in step (c) may be direct, or may be via a linker molecule. The invention also provides a conjugate obtained or obtainable by this process.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase patent application ofPCT/IB2010/002565, filed Sep. 30, 2010, which claims priority to U.S.provisional patent application Ser. No. 61/247,518 filed Sep. 30, 2009,all of which are hereby incorporated by reference in the presentdisclosure in their entirety.

TECHNICAL FIELD

This invention is in the field of conjugating bacterial capsularsaccharides, particularly Staphylococcus aureus type 5 or type 8capsular polysaccharides, to carriers in order to form glycoconjugates.The glycoconjugates are useful for immunisation.

BACKGROUND ART

The capsular saccharides of bacteria have been used for many years invaccines against capsulated bacteria. As saccharides are T-independentantigens, however, they are poorly immunogenic. Conjugation to a carriercan convert T-independent antigens into T-dependent antigens, therebyenhancing memory responses and allowing protective immunity to develop.The most effective saccharide vaccines are therefore based onglycoconjugates, and the prototype conjugate vaccine was againstHaemophilus influenzae type b (‘Hib’) [e.g. see chapter 14 of ref. 97].

Another bacterium for which conjugate vaccines have been described isStaphylococcus aureus (S. aureus). Various polysaccharides have beenisolated from S. aureus for use in glycoconjugates. Two polysaccharidesof particular interest are the type 5 and type 8 capsularpolysaccharides. Approximately 60% of human S. aureus strains are type 8and approximately 30% are type 5. Much of the work on type 5 and type 8conjugates has been performed by Fattom et al., and is described indocuments such as references 1 to 9. The Fattom process for type 5 andtype 8 polysaccharide conjugation typically involves thiolation of apurified polysaccharide using cystamine. The reaction relies on thepresence of carboxylate groups in the capsular polysaccharide. Thesegroups react with cystamine in the presence of a carbodiimide, e.g.EDAC. The derivatised polysaccharide is then conjugated to a carrierprotein such as the Pseudomononas aeruginosa endotoxin A (ETA),typically via a linker [2]. Other researchers have carried outconjugation of purified type 5 and type 8 capsular polysaccharides byreductive amination [10 and 11]; glutaradehyde coupling [10]; orreaction of hydroxyl groups on the polysaccharides with cyanylatingagents like CDAP [12] or cyanuric trichloride [13].

Although conjugate vaccines prepared by the Fattom process have beenshown to be safe and immunogenic in humans [5], there remains a need forfurther and better ways of preparing conjugates of S. aureus type 5 ortype 8 capsular polysaccharides.

DISCLOSURE OF THE INVENTION

The invention is based on a conjugation method that can be used in placeof the conjugation methods disclosed in the prior art. Unlike thesemethods, the method of the invention does not involve conjugation viahydroxyl or carboxylate groups in the polysaccharide. The methodtherefore leaves these groups in a form that is closer than the priorart to the form seen in the native polysaccharide. Instead of usingthese groups, the method involves the generation of reactive aldehydegroups in the polysaccharide for use in conjugation. The resultantconjugates may have different, preferably improved, immunologicalproperties compared to the conjugates of the prior art.

The invention therefore provides alternative or improved methods forconjugating S. aureus type 5 or type 8 capsular polysaccharide to acarrier protein and conjugates obtained therefrom. The invention alsoprovides intermediates that are useful in the methods of the inventionand methods for preparing these intermediates.

In a first aspect, the invention provides a process for preparing aconjugate of a S. aureus type 5 or type 8 capsular polysaccharide and acarrier molecule, comprising the steps of: (a) depolymerising thecapsular polysaccharide, to give a polysaccharide fragment; (b)oxidising the fragment in order to introduce an aldehyde group into atleast one saccharide residue in the fragment, to give an oxidisedsaccharide residue; and (c) coupling the oxidised saccharide residue toa carrier molecule via the aldehyde group, thereby giving the conjugate.The coupling in step (c) may be direct, or it may be via a linkermolecule. The invention also provides a conjugate obtained or obtainableby this process.

The Capsular Polysaccharide

The invention is based on the capsular polysaccharides of S. aureus type5 and type 8. The structures of type 5 and type 8 capsularpolysaccharides were described in references 14 and 15 as:

Type 5

-   -   →4)-β-D-ManNAcA(3OAc)-(1→4)-α-L-FucNAc(1→3)-β-D-FucNAc-(1-→

Type 8

-   -   →3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→

Recent NMR spectroscopy data [16] has led to a revision of thesestructures to:

Type 5

-   -   →4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-(1→

Type 8

-   -   →3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→

The polysaccharide may be chemically modified relative to the capsularpolysaccharide as found in nature.

For example, the polysaccharide may be de-O-acetylated (partially orfully), de-N-acetylated (partially or fully), N-propionated (partiallyor fully), etc. De-acetylation may occur before, during or afterconjugation, but typically occurs before conjugation. Depending on theparticular polysaccharide, de-acetylation may or may not affectimmunogenicity e.g. the NEISVAC-C™ vaccine uses a de-O-acetylatedpolysaccharide, whereas MENJUGATE™ is acetylated, but both vaccines areeffective. The effect of de-acetylation etc. can be assessed by routineassays. For example, the relevance of O-acetylation on S. aureus type 5or type 8 capsular polysaccharides is discussed in reference 6. Thenative polysaccharides are said in this document to have 75%O-acetylation. These polysaccharides induced antibodies to both thepolysaccharide backbone and O-acetyl groups. Polysaccharides with 0%O-acetylation still elicited antibodies to the polysaccharide backbone.Both types of antibody were opsonic against S. aureus strains thatvaried in their O-acetyl content. Accordingly, the type 5 or type 8capsular polysaccharides used in the present invention may have between0 and 100% O-acetylation. For example, the degree of O-acetylation ofthe type 5 capsular polysaccharide may be 10-100%, 10-100%, 20-100%,30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%,60-90%, 70-90% or 80-90%. Alternatively, 0% O-acetylated type 5 capsularpolysaccharide may be used. Similarly, the degree of O-acetylation ofthe type 8 capsular polysaccharide may be 10-100%, 10-100%, 20-100%,30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 50-90%,60-90%, 70-90% or 80-90%. Alternatively, 0% O-acetylated type 8 capsularpolysaccharide may be used. In one embodiment, the degree ofO-acetylation of the type 5 and type 8 capsular polysaccharides may be10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%,90-100%, 50-90%, 60-90%, 70-90% or 80-90%. In other embodiments, 0%O-acetylated type 5 and type 8 capsular polysaccharides are used. Thedegree of N-acetylation of the type 5 capsular polysaccharide used inthe invention may be 0-100%, 50-100%, 75-100%, 80-100%, 90-100%, or95-100%. Typically, the degree of N-acetylation of the type 5 capsularpolysaccharide is 100%. Similarly, the degree of N-acetylation of thetype 8 capsular polysaccharide used in the invention may be 0-100%,50-100%, 75-100%, 80-100%, 90-100%, or 95-100%. Typically, the degree ofN-acetylation of the type 8 capsular polysaccharide is 100%. In oneembodiment, the degree of N-acetylation of the type 5 and type 8capsular polysaccharides may be 0-100%, 50-100%, 75-100%, 80-100%,90-100%, or 95-100%. Typically, the degree of N-acetylation of the type5 and type 8 capsular polysaccharide is 100%.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (e.g. asdescribed in references 17, 18, 19 or 20). A further method is describedin reference 21. Similar methods may be used to determine the degree ofN-acetylation of the polysaccharide. O-acetyl groups may be removed byhydrolysis, for example by treatment with a base such as anhydroushydrazine [22] or NaOH [6]. Similar methods may be used to removeN-acetyl groups. To maintain high levels of O-acetylation on type 5and/or 8 capsular polysaccharides, treatments that lead to hydrolysis ofthe O-acetyl groups are minimised, e.g. treatments at extremes of pH.

Capsular polysaccharides can be purified by known techniques, asdescribed in the references herein. A typical process involvesphenol-ethanol inactivation of S. aureus cells, centrifugation,lysostaphin treatment, RNase/DNase treatment, centrifugation, dialysis,protease treatment, further dialysis, filtration, precipitation withethanol/CaCl₂, dialysis, freeze-drying, anion exchange chromatography,dialysis, freeze-drying, size exclusion chromatography, dialysis andfreeze-drying [1]. An alternative process involves autoclaving S. aureuscells, ultrafiltration of the polysaccharide-containing supernatant,concentration, lyophilisation, treatment with sodium metaperiodate toremove teichoic acid, further ultrafiltration, diafiltration, highperformance size exclusion liquid chromatography, dialysis andfreeze-drying [23]. Preferably, the purification process described inreference 24 is used.

The invention is not limited to polysaccharides purified from naturalsources, however, and the polysaccharides may be obtained by othermethods, such as total or partial synthesis.

The Carrier Molecule

The invention involves the use of carrier molecules, which are typicallyproteins. In general, covalent conjugation of saccharides to carriersenhances the immunogenicity of saccharides as it converts them fromT-independent antigens to T-dependent antigens, thus allowing primingfor immunological memory. Conjugation is particularly useful forpaediatric vaccines [e.g. ref. 25] and is a well known technique [e.g.reviewed in refs. 26 to 34].

Preferred carrier proteins are bacterial toxins, such as diphtheria ortetanus toxins, or toxoids or mutants thereof. The inventors have foundthat the CRM197 diphtheria toxin mutant [35] is suitable. Pseudomonasaeruginosa exotoxin A (ETA) and its non-toxic mutant recombinantexoprotein A (rEPA) have been used as carrier proteins for S. aureustype 5 or type 8 capsular polysaccharides ([1] and [2]). S. aureusα-haemolysin (α-toxin) ([10] and [36]), ovalbumin [13] and human serumalbumin [11] have also been used. These carriers may be used in thepresent invention.

Other suitable carrier proteins include the N. meningitidis outermembrane protein complex [37], synthetic peptides [38,39], heat shockproteins [40,41], pertussis proteins [42,43], cytokines [44],lymphokines [44], hormones [44], growth factors [44], human serumalbumin (typically recombinant), artificial proteins comprising multiplehuman CD4⁺ T cell epitopes from various pathogen-derived antigens [45]such as N19 [46], protein D from H. influenzae [47-49], pneumococcalsurface protein PspA [50], pneumolysin [51] or its non-toxic derivatives[52], iron-uptake proteins [53], toxin A or B from C. difficile [54], aGBS protein [55], a GAS protein [56] etc.

Other suitable carrier proteins include S. aureus protein antigens, forexample the S. aureus protein antigens set out below.

It is possible to use more than one carrier protein e.g. to reduce therisk of carrier suppression. Thus different carrier proteins can be usedfor the type 5 and type 8 capsular polysaccharides, e.g. type 5polysaccharide might be conjugated to CRM197 while type 8 polysaccharidemight be conjugated to rEPA. It is also possible to use more than onecarrier protein for a particular polysaccharide antigen e.g. type 5polysaccharide might be in two groups, with one group conjugated toCRM197 and the other conjugated to rEPA. Typically, however, the samecarrier protein is used for all polysaccharides.

A single carrier protein might carry more than one polysaccharideantigen [57,58]. For example, a single carrier protein might haveconjugated to it type 5 and type 8 capsular polysaccharides. To achievethis goal, different polysaccharides can be mixed prior to theconjugation process. Typically, however, there are separate conjugatesfor each polysaccharide, with the different polysaccharides being mixedafter conjugation. The separate conjugates may be based on the samecarrier.

Depolymerisation

In step (a) of the process of the invention, the capsular polysaccharideis depolymerised to give a polysaccharide fragment. Depolymerisation ofthe type 8 capsular polysaccharide by sonication prior to conjugationhas been reported [3]. The authors concluded that low molecular weighttype 8 was not immunogenic. Although these authors therefore favouredhigh molecular weight polysaccharides, the present inventionsurprisingly makes use of polysaccharide fragments with a lowermolecular weight than native capsular polysaccharides.

Full-length polysaccharides may be depolymerised to give shorterfragments for use in the invention by various methods. The inventorshave found that methods that result in cleavage of (1→3) glycosidiclinkages between the α-L-FucNAc(3OAc) and β-D-FucNAc residues in thetype 5 capsular polysaccharide are particularly suitable. When thesemethods are applied to the type 5 capsular polysaccharide, they resultin a polysaccharide fragment having a β-D-FucNAc-(1→moiety at itsnon-reducing terminus. This moiety includes two vicinal hydroxyl groups.Similarly, when these methods are applied to the type 8 capsularpolysaccharide, they are thought to result in a polysaccharide fragmenthaving an α-D-FucNAc-(1→moiety at is non-reducing terminus, which moietyalso includes two vicinal hydroxyl groups. The vicinal hydroxyl groupsin the type 5 or type 8 polysaccharide fragment provide a handle forsubsequent conjugation of the fragment to a carrier molecule, asdescribed below.

Accordingly, in a further aspect the invention provides a process fortreating a S. aureus type 5 capsular polysaccharide comprising the stepof depolymerising the capsular polysaccharide, to give a polysaccharidefragment having a β-D-FucNAc-(1→moiety at its non-reducing terminus. Ina related aspect, the invention provides a process for treating a S.aureus type 8 capsular polysaccharide comprising the step ofdepolymerising the capsular polysaccharide, to give a polysaccharidefragment having an α-D-FucNAc-(1→moiety at its non-reducing terminus.The capsular polysaccharide may be a S. aureus type 5 or type 8 capsularpolysaccharide as described in “The capsular polysaccharide” above. Theinvention also provides a polysaccharide fragment obtained or obtainableby either of these processes.

The inventors have found that the depolymerisation may be carried out byacid hydrolysis. For acid hydrolysis, it is preferred to use a mildacid, e.g. acetic acid, to avoid side-reactions at other groups withinthe polysaccharide. The skilled person would be capable of identifyingsuitable acids and conditions (e.g. of concentration, temperature and/ortime) for hydrolysis. For example, the inventors have found thattreatment of polysaccharide at 2 mg/ml with 2% acetic acid (v/v) at 90°C. for 3 hours is suitable. The inventors have also found that treatmentat 2 mg/ml with 5% acetic acid at 90° C. for 30 minutes, 5 or 6 hours issuitable. Treatment with other acids, e.g. trifluoroacetic or otherorganic acids, may also be suitable. In particular, the inventors havefound that depolymerisation efficiency may be increased, particularlyfor type 8 capsular polysaccharide, by using hydrochloric acid. Forexample, the inventors have found that treatment of polysaccharide with2M hydrochloric acid at 100° C. for 30 minutes is suitable. Theinventors have also found that treatment with 2M hydrochloric acid at100° C. for 1, 1.5, 2 or 2.5 hours is suitable. Such treatment withhydrochloric acid may result in de-O-acetylation of the polysaccharide,e.g. as described below.

Other methods for depolymerisation of the polysaccharide may besuitable. These methods include heating, microfluidisation [59], sonicradiation [3], oxidation-reduction [60] or ozonolysis [61].

Polysaccharide fragments can be identified by chromatography, e.g. sizeexclusion chromatography. Specific molecular masses can be measured bygel filtration relative to pullulan standards, such as those availablefrom Polymer Standard Service [62]. Typically, the fragment of theinvention is a mixture of fragments with masses within a range ofvalues. For the depolymerised type 5 capsular polysaccharide, themolecular mass of the fragment typically varies between 1-500 kDa, e.g.between 5 and 100 kDa, particularly between 10 and 50 kDa and moreparticularly between 20 and 30 kDa. Similarly, for the depolymerisedtype 8 capsular polysaccharide, the molecular mass of the fragment mayvary between 1-500 kDa, e.g. between 5 and 100 kDa, particularly between10 and 50 kDa and more particularly between 20 and 30 kDa. In someembodiments, low molecular weight type 5 and/or type 8 polysaccharidefragments are selected for use in the invention. For example, gelfiltration fractions corresponding to low molecular weight fragments maybe selected and pooled. The low molecular weight polysaccharidefragments typically have a molecular mass that varies between 5 and 20kDa.

The depolymerisation may result in a change to the degree ofO-acetylation of the capsular polysaccharide. For the example, theinventors have found that acid hydrolysis may result in a decrease inthe degree of O-acetylation. In some embodiments, the degree ofO-acetylation of the fragment may be 10-90%, 20-70%, 30-50%,particularly 35-45%. In other embodiments, the degree of O-acetylationof the fragment may be 0-10%, 0-5%, 0-2%, particularly 0%.

Introduction of an Aldehyde Group

In step (b) of the process, the fragment is oxidised in order tointroduce an aldehyde group into at least one saccharide residue in thefragment. This step may involve the introduction of more than onealdehyde group into the saccharide residue. In particular, two aldehydegroups may be introduced. For example, when the depolymerisation in step(a) results in a type 5 polysaccharide fragment having aβ-D-FucNAc-(1→moiety at its non-reducing terminus, the two vicinalhydroxyl groups in this moiety may be oxidised in order to introduce twoaldehyde groups into the moiety. In this way, the β-D-FucNAc-(1→moietymay be the saccharide residue of step (b). Similarly, when thedepolymerisation results in a type 8 polysaccharide fragment having anα-D-FucNAc-(1→moiety at is non-reducing terminus, the two vicinalhydroxyl groups in this moiety may be oxidised to introduce two aldehydegroups. In this way, the α-D-FucNAc-(1→moiety may be the saccharideresidue of step (b).

Accordingly, in a further aspect the invention provides a process forproviding a S. aureus type 5 capsular polysaccharide derivativecomprising the step of oxidising a S. aureus type 5 capsularpolysaccharide having a β-D-FucNAc-(1→moiety at its non-reducingterminus to convert two vicinal hydroxyl groups in theβ-D-FucNAc-(1→moiety into two aldehyde groups. In a related aspect, theinvention provides a process for providing a S. aureus type 8 capsularpolysaccharide derivative comprising the step of oxidising a S. aureustype 8 capsular polysaccharide having an α-D-FucNAc-(1→moiety at isnon-reducing terminus to convert two vicinal hydroxyl groups in theα-D-FucNAc-(1→moiety into two aldehyde groups. The capsularpolysaccharide may be a polysaccharide fragment as described in“Depolymerisation” above. The invention also provides a S. aureuscapsular polysaccharide derivative obtained or obtainable by either ofthese processes.

Typical reactions to produce aldehydes include the use of periodatesalts, and particularly meta-periodates (e.g. sodium or potassiummeta-periodate e.g. NaIO₄), to oxidise vicinal hydroxyl groups [63]. Theskilled person would be capable of identifying suitable conditions foroxidation. For example, the inventors have found that treatment ofpolysaccharide at 2 mg/ml with NaIO₄ at a 1:1 ratio (w/w) at roomtemperature for 1-2 hours in the dark is suitable. The inventors havealso found that treatment of polysaccharide at 2 mg/ml with 93 mM NaIO₄at room temperature for 8 hours in the dark is suitable. Other oxidationconditions can be used, e.g. use of osmium tetroxide, etc.

Coupling to a Carrier Molecule

The coupling of the oxidised saccharide residue to the carrier moleculein step (c) of the process may be direct or via a linker. Any suitableconjugation reaction can be used, with any suitable linker if desired.

When the oxidation in step (b) results in a type 5 polysaccharidefragment having a β-D-FucNAc-(1→moiety at its non-reducing terminus inwhich two aldehyde groups have been introduced into the moiety, thecoupling in step (c) may be via one of these aldehyde groups. In thisway, the oxidised β-D-FucNAc-(1→moiety may be the oxidised saccharideresidue of step (c). Similarly, when the oxidation results in a type 8polysaccharide fragment having an α-D-FucNAc-(1→moiety at isnon-reducing terminus in which two aldehyde groups have been introducedinto the moiety, the coupling in step (c) may be via one of thesealdehyde groups. In this way, the oxidised α-D-FucNAc-(1→moiety may bethe oxidised saccharide residue of step (c).

Accordingly, in a further aspect the invention provides a process forproviding a conjugated S. aureus type 5 capsular polysaccharidecomprising the step of coupling to a carrier molecule a S. aureus type 5capsular polysaccharide having a O-D-FucNAc-(1→moiety at itsnon-reducing terminus that has been oxidised to convert two vicinalhydroxyl groups into two aldehyde groups, wherein the coupling is viaone of the aldehyde groups. In a related aspect, the invention providesa process for providing a conjugated S. aureus type 8 capsularpolysaccharide comprising the step of coupling to a carrier molecule aS. aureus type 8 capsular polysaccharide having an α-D-FucNAc-(1→moietyat is non-reducing terminus that has been oxidised to convert twovicinal hydroxyl groups into two aldehyde groups, wherein the couplingis via one of the aldehyde groups. The capsular polysaccharide may be acapsular polysaccharide as described in “Introduction of an aldehydegroup” above. The carrier molecule may be a carrier as described in “Thecarrier molecule” above. The invention also provides a conjugatedcapsular polysaccharide obtained or obtainable by either of theseprocesses.

Attachment of the oxidised saccharide residue or linker to the carrieris typically via an amine (—NH₂) group e.g. in the side chain of alysine or residue in a carrier protein, or of an arginine residue.Attachment to the carrier may also be via a sulphydryl (—SH) group e.g.in the side chain of a cysteine residue. The inventors have found thatdirect coupling may be conveniently achieved by reacting an aldehydegroup in the oxidised saccharide residue with an amine group in thecarrier by reductive amination. Direct coupling of this nature istherefore preferred in the present invention. In contrast, reference 2suggests that linkers may be advantageous in S. aureus type 5 and 8conjugates. If desired, coupling via a linker may be used in the presentinvention, e.g. by reacting an aldehyde group in the oxidised saccharideresidue with an amine group in the linker by reductive amination, or byconverting the aldehyde group into an amine group by reductive aminationto provide an amine group for attachment of the linker.

Reductive amination is a standard technique in organic chemistry, andhas been used extensively in the production of conjugates of capsularpolysaccharides for vaccine use, including S. aureus capsularpolysaccharides [10]. In one embodiment, an aldehyde group in theoxidised saccharide residue reacts with an amine group in the carrier orlinker. This can conveniently be achieved by combining thepolysaccharide with the carrier or linker in the presence of anappropriate reducing agent (e.g. cyanoborohydrides, such as sodiumcyanoborohydride NaBH₃CN; borane-pyridine; sodium triacetoxyborohydride;borohydride exchange resin; etc.). In another embodiment, an aldehydegroup is converted into an amine group by reductive amination to providean amine group for attachment of the linker. The reductive aminationinvolves either ammonia or a primary amine (NH₂R). This can convenientlybe achieved by using an ammonium salt (e.g. ammonium chloride) incombination with an appropriate reducing agent (e.g. as listed above).The skilled person would be capable of identifying suitable conditionsfor reductive amination. For example, the inventors have found thattreatment of polysaccharide at 10 mg/ml with carrier protein at a 4:1polysaccharide:protein ratio (w/w) and NaBH₃CN at a 2:1polysaccharide:NaBH₃CN ratio is suitable.

Coupling via a linker group may be made using any known procedure, e.g.the reductive amination procedures described above. In one embodiment, abifunctional linker may be used to provide a first group for coupling tothe aldehyde group in the oxidised saccharide residue and a second groupfor coupling to the carrier. For example, a bifunctional linker of theformula X₁-L-X₂ may be used, where X₁ can react with the aldehyde; X₂can react with the carrier; and L is a linking moiety in the linker. Atypical X₁ group is an amine group. Typical L groups are straight chainalkyls with 1 to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈,C₉, C₁₀) e.g. —(CH₂)₄— or —(CH₂)₃—. In another embodiment, abifunctional linker may be used to provide a first group for coupling toan amine group derived from the aldehyde group in the oxidisedsaccharide residue (e.g. by reductive amination as described above) anda second group for coupling to the carrier (typically for coupling to anamine in the carrier). For example, a homobifunctional linker of theformula X-L-X may be used, where the two X groups are the same as eachother and can react with the amines; and where L is a linking moiety inthe linker. A typical X group is N-oxysuccinimide. L typically hasformula -L′-L²-L′-, where L′ is carbonyl. Typical L² groups are straightchain alkyls with 1 to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇,C₈, C₉, C₁₀) e.g. —(CH₂)₄—. A typical linker is thus adipic acidN-hydroxysuccinimide diester (SIDEA):

Other X groups are those which form esters when combined with HO-L-OH,such as norborane, p-nitrobenzoic acid, and sulfo-N-hydroxysuccinimide.Further bifunctional linkers reactive with amines for use with theinvention include acryloyl halides (e.g. chloride) [65], haloacylhalides[66], disuccinimidyl glutarate, disuccinimidyl suberate, ethylene glycolbis[succinimidylsuccinate], etc.

The linker will generally be added in molar excess to thepolysaccharide. The linker/polysaccharide reaction will generally takeplace in an aprotic solvent (e.g. DMSO, ethanol acetate, etc.), as thelinkers are typically insoluble in water. Where water-soluble linkersare used, however, then a wider range of solvents is available,including protic solvents such as water. Suitable linkers includesulphonated forms, such as sulphonated SIDEA:

When a linker is used, the conjugate will comprise a linker moiety. Thismoiety originates neither in the polysaccharide nor the carrier, but isa third molecule used during conjugate preparation, and can readily bedistinguished from both the polysaccharide and carrier protein in afinal conjugate product. The linker moiety may include atoms such ascarbon, hydrogen, oxygen and/or nitrogen. Linkers that comprise carbonand hydrogen are typical, and linkers that further comprise oxygenand/or nitrogen are also typically used. Linkers that include nitrogenatoms may include a carbon atom bonded to a nitrogen atom, which in turnis bonded to a second carbon atom (—C—N—C—). Linkers that include anoxygen atom typically include it as part of a carbonyl group. Linkermoieties with a molecular weight of between 30-500 Da are typical.Linkers containing two carbonyl groups are also typical.

A particularly useful linker moiety is —NH—C(O)—(CH₂)_(n)—C(O)—, whereinn is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The value of n is typically 4. Theterminal —NH— in this linker is usually attached to a carbon atom fromthe polysaccharide moiety. The terminal —C(O)— is usually attached to anitrogen atom in an amino acid side chain in the carrier. A preferredlinker moiety can conveniently be introduced by a process involving:reductive amination of the aldehyde in the oxidised saccharide residue;reaction of the resulting —NH₂ group with a bifunctional linker that isa diester (e.g. a disuccinimidyl ester) of a dioic acid (e.g. of adipicacid, HOOC—(CH₂)₄—COOH); and reductive amination of the product (seeFIG. 6 [64]).

Other chemistries that can be used to attach a linker to a —NH₂ group inthe polysaccharide, include:

-   -   acryloylation (e.g. by reaction with acryloyl chloride),        followed by Michael-type addition to either the ε-NH₂ of an        amino acid side chain or to a —SH of a cysteine side chain [65].        The resulting linker is —NH—C(O)—(CH₂)₂— (propionamido).    -   reaction with a haloacylhalide, followed by reaction with the        ε-NH₂ of an amino acid side chain or to a —SH of a cysteine side        chain [66]. The linker is —NH—C(O)—CH₂—.

Conjugates with a polysaccharide:protein ratio (w/w) of between 1:20(i.e. excess protein) and 20:1 (i.e. excess polysaccharide) aretypically produced by the method of the invention. Ratios of 1:10 to 1:1are preferred, particularly ratios between 1:5 and 1:2 and, mostpreferably, about 1:3. In contrast, type 5 and type 8 capsularpolysaccharide conjugates made by processes of the prior art tend tohave higher ratios, e.g. between 0.73:1 and 1.08:1 in references 1, 2and 3. In particular embodiments of the invention, thepolysaccharide:protein ratio (w/w) for type 5 capsular polysaccharideconjugate is between 1:10 and 1:2; and/or the polysaccharide:proteinratio (w/w) for type 8 capsular polysaccharide conjugate is between 1:5and 7:10.

Compositions may include a small amount of free carrier [67]. When agiven carrier protein is present in both free and conjugated form in acomposition of the invention, the unconjugated form is preferably nomore than 5% of the total amount of the carrier protein in thecomposition as a whole, and more preferably present at less than 2% byweight.

After conjugation, free and conjugated polysaccharides can be separated.There are many suitable methods, including hydrophobic chromatography,tangential ultrafiltration, diafiltration etc. [see also refs. 68 & 69,etc.].

Combinations of Conjugates and Other Antigens

As well as providing individual conjugates as described above, theinvention provides a composition comprising a conjugate of the inventionand one or more further antigens. The composition is typically animmunogenic composition.

The further antigen(s) may comprise further conjugates of the invention,and so the invention provides a composition comprising more than oneconjugate of the invention. In particular, the present inventionprovides a composition comprising a type 5 capsular polysaccharideconjugate of the invention and a type 8 capsular polysaccharideconjugate of the invention. Alternatively, the further antigen(s) may betype 5 or type 8 capsular polysaccharide conjugates prepared by methodsother than those of the invention, e.g. the methods of references 1 to13 above. The further antigen(s) may similarly be type 5 or type 8capsular polysaccharide conjugates prepared by the methods of references59, 70, 71, 72, 73, and 74, and particularly the exemplified methods inthose documents. Accordingly, the invention provides a compositioncomprising a type 5 capsular polysaccharide conjugate and a type 8capsular polysaccharide conjugate, wherein one of the conjugates (thetype 5 conjugate or the type 8 conjugate) is a conjugate of theinvention and the other conjugate is not a conjugate of the invention.

The further antigen(s) may comprise other S. aureus antigens, includingprotein and saccharide antigens, as set out below.

The further antigen(s) may comprise antigens from non-S. aureuspathogens. Thus the compositions of the invention may further compriseone or more non-S. aureus antigens, including additional bacterial,viral or parasitic antigens. These may be selected from the following:

-   -   a protein antigen from N. meningitidis serogroup B, such as        those in refs. 75 to 81, with protein ‘287’ (see below) and        derivatives (e.g. ‘ΔG287’) being particularly useful.    -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. 82, 83, 84, 85        etc.    -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 86 from        serogroup C or the oligosaccharides of ref. 87.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. refs.        88-90; chapters 22 & 23 of ref. 97].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 91, 92; chapter 15 of ref. 97].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 92,93; chapter 16 of ref. 97].    -   an antigen from hepatitis C virus [e.g. 94].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 95 & 96; chapter 21 of ref.        97].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        13 of ref. 97].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 27 of        ref. 97].    -   a saccharide antigen from Haemophilus influenzae B [e.g. chapter        14 of ref. 97]    -   an antigen from N. gonorrhoeae [e.g. 75, 76, 77].    -   an antigen from Chlamydia pneumoniae [e.g. 98, 99, 100, 101,        102, 103, 104].    -   an antigen from Chlamydia trachomatis [e.g. 105].    -   an antigen from Porphyromonas gingivalis [e.g. 106].    -   polio antigen(s) [e.g. 107, 108; chapter 24 of ref. 97] such as        IPV.    -   rabies antigen(s) [e.g. 109] such as lyophilised inactivated        virus [e.g. 110, RabAvert™].    -   measles, mumps and/or rubella antigens [e.g. chapters 19, 20 and        26 of ref. 97].    -   influenza antigen(s) [e.g. chapters 17 & 18 of ref. 97], such as        the haemagglutinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. 111].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 112, 113, 114].    -   an antigen from Streptococcus agalactiae (group B streptococcus)        [e.g. 56, 115-117].    -   an antigen from S. epidermidis [e.g. type I, II and/or III        capsular polysaccharide obtainable from strains ATCC-31432,        SE-360 and SE-10 as described in refs. 118, 119 and 120.

Where a saccharide or carbohydrate antigen is used, it is typicallyconjugated to a carrier in order to enhance immunogenicity. Conjugationof H. influenzae B, meningococcal and pneumococcal saccharide antigensis well known.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[96]).

Where a diphtheria antigen is included in the composition it is typicalalso to include tetanus antigen and pertussis antigens. Similarly, wherea tetanus antigen is included it is typical also to include diphtheriaand pertussis antigens. Similarly, where a pertussis antigen is includedit is typical also to include diphtheria and tetanus antigens.

Antigens may be adsorbed to an aluminium salt.

One type of preferred composition includes further antigens that affectthe immunocompromised, and so the S. aureus conjugates of the inventioncan be combined with one or more antigens from the following non-S.aureus pathogens: Steptococcus agalactiae, Staphylococcus epidermis,influenza virus, Enterococcus faecalis, Pseudomonas aeruginosa,Legionella pneumophila, Listeria monocytogenes, Neisseria meningitidis,and parainfluenza virus.

Another type of preferred composition includes further antigens frombacteria associated with nosocomial infections, and so the S. aureusconjugates of the invention can be combined with one or more antigensfrom the following non-S. aureus pathogens: Clostridium difficile;Pseudomonas aeruginosa; Candida albicans; and extraintestinal pathogenicEscherichia coli.

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 121to 129]. Protein components of the compositions of the invention maythus be replaced by nucleic acid (usually DNA e.g. in the form of aplasmid) that encodes the protein.

In practical terms, there may be an upper limit to the number ofantigens included in compositions of the invention. The number ofantigens (including S. aureus antigens) in a composition of theinvention may be less than 20, less than 19, less than 18, less than 17,less than 16, less than 15, less than 14, less than 13, less than 12,less than 11, less than 10, less than 9, less than 8, less than 7, lessthan 6, less than 5, less than 4, or less than 3. The number of S.aureus antigens in a composition of the invention may be less than 6,less than 5, or less than 4.

Pharmaceutical Compositions and Methods

The invention provides a pharmaceutical composition comprising (a) aconjugate of the invention and (b) a pharmaceutically acceptablecarrier. Typical ‘pharmaceutically acceptable carriers’ include anycarrier that does not itself induce the production of antibodies harmfulto the individual receiving the composition. Suitable carriers aretypically large, slowly metabolised macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, sucrose [130], trehalose [131], lactose,and lipid aggregates (such as oil droplets or liposomes). Such carriersare well known to those of ordinary skill in the art. The vaccines mayalso contain diluents, such as water, saline, glycerol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in reference 132.

Compositions of the invention may be in aqueous form (i.e. solutions orsuspensions) or in a dried form (e.g. lyophilised). If a dried vaccineis used then it will be reconstituted into a liquid medium prior toinjection. Lyophilisation of conjugate vaccines is known in the art e.g.the MENJUGATE™ product is presented in lyophilised form, whereasNEISVAC-C™ and MENINGITEC™ are presented in aqueous form. To stabiliseconjugates during lyophilisation, it may be typical to include a sugaralcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose)e.g. at between 1 mg/ml and 30 mg/ml (e.g. about 25 mg/ml) in thecomposition.

Compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses.

Aqueous compositions of the invention are also suitable forreconstituting other vaccines from a lyophilised form. Where acomposition of the invention is to be used for such extemporaneousreconstitution, the invention provides a kit, which may comprise twovials, or may comprise one ready-filled syringe and one vial, with thecontents of the syringe being used to reactivate the contents of thevial prior to injection.

Compositions of the invention may be packaged in unit dose form or inmultiple dose form. For multiple dose forms, vials are preferred topre-filled syringes. Effective dosage volumes can be routinelyestablished, but a typical human dose of the composition has a volume of0.5 ml e.g. for intramuscular injection.

The pH of the composition is typically between 6 and 8, e.g. about 7.Stable pH may be maintained by the use of a buffer. If a compositioncomprises an aluminium hydroxide salt, it is typical to use a histidinebuffer [133]. The composition may be sterile and/or pyrogen-free.Compositions of the invention may be isotonic with respect to humans.

Compositions of the invention are immunogenic, and are more preferablyvaccine compositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic. Immunogenic compositionsused as vaccines comprise an immunologically effective amount ofantigen(s), as well as any other components, as needed. By‘immunologically effective amount’, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, age, the taxonomic group of individual to be treated (e.g.non-human primate, primate, etc.), the capacity of the individual'simmune system to synthesise antibodies, the degree of protectiondesired, the formulation of the vaccine, the treating doctor'sassessment of the medical situation, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials.

Within each dose, the quantity of an individual saccharide antigen willgenerally be between 1-50 μg (measured as mass of saccharide) e.g. about1 μg, about 2.5 μg, about 4 μg, about 5 μg, or about 10 μg.

S. aureus affects various areas of the body and so the compositions ofthe invention may be prepared in various forms. For example, thecompositions may be prepared as injectables, either as liquid solutionsor suspensions. The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as spray,drops, gel or powder [e.g. refs 134 & 135]. Success with nasaladministration of pneumococcal saccharides [136,137], Hib saccharides[138], MenC saccharides [139], and mixtures of Hib and MenC saccharideconjugates [140] has been reported.

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format.

Compositions of the invention may comprise detergent e.g. a TWEEN®(polysorbate), such as TWEEN® 80. Detergents are generally present atlow levels e.g. <0.01%.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical.

Compositions of the invention will generally include a buffer. Aphosphate buffer is typical.

Compositions of the invention will generally be administered inconjunction with other immunoregulatory agents. In particular,compositions will usually include one or more adjuvants. Such adjuvantsinclude, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 141], or mixtures ofdifferent mineral compounds (e.g. a mixture of a phosphate and ahydroxide adjuvant, optionally with an excess of the phosphate), withthe compounds taking any suitable form (e.g. gel, crystalline,amorphous, etc.), and with adsorption to the salt(s) being typical. Themineral containing compositions may also be formulated as a particle ofmetal salt [142].

Aluminum salts may be included in vaccines of the invention such thatthe dose of Al³⁺ is between 0.2 and 1.0 mg per dose.

A typical aluminium phosphate adjuvant is amorphous aluminiumhydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92, includedat 0.6 mg Al³⁺/ml. Adsorption with a low dose of aluminium phosphate maybe used e.g. between 50 and 100 μg Al³⁺ per conjugate per dose. Where analuminium phosphate it used and it is desired not to adsorb an antigento the adjuvant, this is favoured by including free phosphate ions insolution (e.g. by the use of a phosphate buffer).

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 (5% Squalene, 0.5% TWEEN®80, and 0.5% SPAN® 85, formulated into submicron particles using amicrofluidizer) [Chapter 10 of ref. 141; see also refs. 143-145]. MF59is used as the adjuvant in the FLUAD™ influenza virus trivalent subunitvaccine.

Particularly useful adjuvants for use in the compositions are submicronoil-in-water emulsions. Preferred submicron oil-in-water emulsions foruse herein are squalene/water emulsions optionally containing varyingamounts of MTP-PE, such as a submicron oil-in-water emulsion containing4-5% w/v squalene, 0.25-1.0% w/v TWEEN® 80 (polyoxyelthylenesorbitanmonooleate), and/or 0.25-1.0% SPAN® 85 (sorbitan trioleate), and,optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphosphoryloxy)-ethylamine(MTP-PE). Submicron oil-in-water emulsions, methods of making the sameand immunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in references 143 & 146-147.Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

C. Saponin Formulations [Chapter 22 of Ref 141]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponins isolated from thebark of the Quillaia saponaria Molina tree have been widely studied asadjuvants. Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref.148. Saponin formulations may also comprise a sterol, such ascholesterol [149].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs) [chapter 23 ofref. 141]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA and QHC. ISCOMs are further described in refs. 149-151. Optionally,the ISCOMS may be devoid of additional detergent(s) [152].

A review of the development of saponin based adjuvants can be found inrefs. 153 & 154.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 155-160. Virosomes are discussed further in, for example, ref.161

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 162. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [162]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [163,164].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 165 & 166.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 167, 168 and 169 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 170-175.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [176]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 177-179. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 176 & 180-182.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 183 and as parenteraladjuvants in ref. 184. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 185-192. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref 193, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [194], etc.) [195], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [196] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [197].

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref. 141)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 198-200.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [201]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [202] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [203]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

K. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 204 and 205.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “RESIQUIMOD 3M”),described further in refs. 206 and 207.

N. Thiosemicarbazone Compounds.

Examples of thiosemicarbazone compounds, as well as methods offormulating, manufacturing, and screening for compounds all suitable foruse as adjuvants in the invention include those described in ref. 208.The thiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

O. Tryptanthrin Compounds.

Examples of tryptanthrin compounds, as well as methods of formulating,manufacturing, and screening for compounds all suitable for use asadjuvants in the invention include those described in ref. 209. Thetryptanthrin compounds are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the followingcombinations may be used as adjuvant compositions in the invention: (1)a saponin and an oil-in-water emulsion [210]; (2) a saponin (e.g.QS21)+a non-toxic LPS derivative (e.g. 3dMPL) [211]; (3) a saponin (e.g.QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol; (4) asaponin (e.g. QS21)+3dMPL+IL-12 (optionally +a sterol) [212]; (5)combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions [213]; (6) SAF, containing 10% squalane, 0.4% TWEEN® 80, 5%PLURONIC®-block polymer L121, and thr-MDP, either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion. (7) RIBI™ adjuvant system (RAS), (Ribi Immunochem) containing2% squalene, 0.2% TWEEN® 80, and one or more bacterial cell wallcomponents from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (DETOX™); and (8) one or more mineral salts (such as an aluminumsalt)+a non-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 141.

The use of aluminium salt adjuvants is particularly useful, and antigensare generally adsorbed to such salts. The MENJUGATE™ and NEISVAC™conjugates use a hydroxide adjuvant, whereas MENINGITEC™ uses aphosphate adjuvant. It is possible in compositions of the invention toadsorb some antigens to an aluminium hydroxide but to have otherantigens in association with an aluminium phosphate. Typically, however,only a single salt is used, e.g. a hydroxide or a phosphate, but notboth. Not all conjugates need to be adsorbed i.e. some or all can befree in solution.

Methods of Treatment

The invention also provides a method for raising an immune response in amammal, comprising administering a pharmaceutical composition of theinvention to the mammal. The immune response is preferably protectiveand preferably involves antibodies. The method may raise a boosterresponse.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc. A preferred class of humans for treatment are patients at risk ofnosocomial infection, particularly those with end-stage renal diseaseand/or on haemodialysis. Other patients at risk of nosocomial infectionare also preferred, e.g. immunodeficient patients or those who haveundergone surgery, especially cardiac surgery, or trauma. Anotherpreferred class of humans for treatment are patients at risk ofbacteremia. A further preferred class are patients suffering from orpreviously exposed to influenza virus, as S. aureus has been linked withpost-infection pneumonia in these patients.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of a conjugate of the invention inthe manufacture of a medicament for raising an immune response in amammal.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by S. aureus, e.g. skin infections, suchas impetigo, boils, cellulitis folliculitis, styes, furuncles,carbuncles, scalded skin syndrome and abscesses, septic arthritis,pneumonia, mastitis, phlebitis, meningitis, urinary tract infections,osteomyelitis, endocarditis, toxic shock syndrome (TSS), septicaemia andnosocomial infections.

One way of checking efficacy of therapeutic treatment involvesmonitoring S. aureus infection after administration of the compositionof the invention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the S. aureus antigensafter administration of the composition.

Preferred compositions of the invention can confer an antibody titre ina patient that is superior to the criterion for seroprotection for eachantigenic component for an acceptable percentage of human subjects.Antigens with an associated antibody titre above which a host isconsidered to be seroconverted against the antigen are well known, andsuch titres are published by organisations such as WHO. Preferably morethan 80% of a statistically significant sample of subjects isseroconverted, more preferably more than 90%, still more preferably morethan 93% and most preferably 96-100%.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

S. aureus Antigens

As mentioned above, one or more further S. aureus antigens can beincluded in compositions of the invention. The antigens may be proteinor saccharide antigens. S. aureus protein antigens may be used ascarrier proteins for conjugates of the invention, carrier proteins forother conjugates, or as unconjugated protein antigens. S. aureussaccharide antigens may be used as the saccharides for other conjugateor as unconjugated saccharide antigens.

Suitable S. aureus saccharide antigens include the exopolysaccharide ofS. aureus, which is a poly-N-acetylglucosamine (PNAG). Thispolysaccharide is present in both S. aureus and S. epidermidis and canbe isolated from either source [214,215]. For example, PNAG may beisolated from S. aureus strain MN8m [216]. The saccharide antigen may bea polysaccharide having the size that arises during purification of theexopolysaccharide from bacteria, or it may be an polysaccharide achievedby fragmentation of such a polysaccharide e.g. size can vary from over400 kDa to between 75 and 400 kDa, or between 10 and 75 kDa, or up to 30repeat units. The saccharide antigen can have various degrees ofN-acetylation and, as described in reference 217, the PNAG may be lessthan 40% N-acetylated (e.g. less than 35, 30, 20, 15, 10 or 5%N-acetylated; deacetylated PNAG is also known as dPNAG). Deacetylatedepitopes of PNAG can elicit antibodies that are capable of mediatingopsonic killing. The preparation of dPNAG is described in reference 218.The PNAG may or may not be O-succinylated e.g. it may be O-succinylatedon fewer less than 25, 20, 15, 10, 5, 2, 1 or 0.1% of residues. The PNAGmay be conjugated to a carrier molecule as described above oralternatively unconjugated.

Another suitable S. aureus saccharide antigen is the type 336 antigen,which is a β-linked hexosamine with no O-acetylation [219,220]. The type336 antigen is cross-reactive with antibodies raised against the 336strain (ATCC 55804). The type 336 antigen may be conjugated to a carriermolecule as described above or alternatively unconjugated.

Suitable S. aureus protein antigens include the following S. aureusantigens (or antigens comprising immunogenic fragment(s) thereof) [e.g.see references 221-228]: AhpC, AhpF, Autolysin amidase, Autolysinglucosaminidase, Collagen binding protein CAN, EbhB, GehD lipase,Heparin binding protein HBP (17 kDa), Laminin receptor, MAP, MntC (alsoknown as SitC), MRPII, Npase, ORF0594, ORF0657n, ORF0826, PBP4, RAP (RNAIII activating protein), Sai-1, SasK, SBI, SdrG, SdrH, SSP-1, SSP-2 andVitronectin-binding protein.

Further suitable S. aureus protein antigens include a clfA antigen; aclfB antigen; a sdrE2 antigen; a sdrC antigen; a sasF antigen, a empantigen; a sdrD antigen; a spa antigen; a esaC antigen; a esxA antigen;a esxB antigen; a sta006 antigen; a isdC antigen; a Hla antigen; asta011 antigen; a isdA antigen; a isdB antigen; and a sta073 antigen, asdescribed below. One or more (i.e. 1, 2, 3, 4, 5, 6 or more) of theseantigens may be present in a composition of the invention. Of theseantigens, the use of one or more (i.e. 1, 2, 3, 4, 5, 6 or more) of aesxA antigen; a esxB antigen; a sta006 antigen; a Hla antigen; a sta011antigen; and/or a sta073 antigen is specifically envisaged.

For example, a composition of the invention may further comprise one ofthe following combinations of S. aureus protein antigens:

-   -   (1) A esxA antigen, a esxB antigen, a sta006 antigen and a Hla        antigen. The esxA and esxB antigens can usefully be combined as        a hybrid polypeptide, as discussed below, e.g. a EsxAB hybrid        with a esxB antigen downstream of a esxA antigen. The Hla        antigen may be a detoxified mutant e.g. including a H35L        mutation.    -   (2) A esxA antigen, a esxB antigen, a sta006 antigen and a        sta011 antigen. The esxA and esxB antigens may be combined as a        hybrid polypeptide, as discussed below, e.g. an EsxAB hybrid.    -   (3) A esxA antigen, a esxB antigen and a sta011 antigen. The        esxA and esxB antigens can usefully be combined as a hybrid        polypeptide, as discussed below, e.g. a EsxAB hybrid.    -   (4) A esxA antigen, a esxB antigen, a Hla antigen, a sta006        antigen and a sta011 antigen. The esxA and esxB antigens may be        combined as a hybrid polypeptide, as discussed below, e.g. an        EsxAB hybrid. The Hla antigen may be a detoxified mutant e.g.        including a H35L mutation.    -   (5) A esxA antigen, a esxB antigen and a Hla antigen. The esxA        and esxB antigens can usefully be combined as a hybrid        polypeptide, as discussed below, e.g. a EsxAB hybrid. The Hla        antigen may be a detoxified mutant e.g. including a H35L        mutation.    -   (6) A Hla antigen, a sta006 antigen and a sta011 antigen. The        Hla antigen may be a detoxified mutant e.g. including a H35L        mutation.    -   (7) A esxA antigen and a esxB antigen. The esxA and esxB        antigens can usefully be combined as a hybrid polypeptide, as        discussed below, e.g. an EsxAB hybrid.    -   (8) A esxA antigen, a esxB antigen and a sta006 antigen. The        esxA and esxB antigens can usefully be combined as a hybrid        polypeptide, as discussed below, e.g. a EsxAB hybrid.    -   (9) A esxA antigen, a esxB antigen, a sta011 antigen and a        sta073 antigen. The esxA and esxB antigens may be combined as a        hybrid polypeptide, as discussed below, e.g. an EsxAB hybrid.    -   (10) A sta006 antigen and a sta011 antigen.

Further Staphylococcus aureus antigens are disclosed in reference 229.

clfA

The ‘clfA’ antigen is annotated as ‘clumping factor A’. In the NCTC 8325strain clfA is SAOUHSC_(—)00812 and has amino acid sequence SEQ ID NO: 1(GI:88194572). In the Newman strain it is nwmn_(—)0756 (GI:151220968).

Useful clfA antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 1 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 1; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 1, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These clfA proteins include variants of SEQ ID NO: 1.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 1. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 1 while retaining at least one epitopeof SEQ ID NO: 1. The final 368 C-terminal amino acids of SEQ ID NO: 1can usefully be omitted. The first 39 N-terminal amino acids of SEQ IDNO: 1 can usefully be omitted. Other fragments omit one or more proteindomains.

SEQ ID NO: 2 is a useful fragment of SEQ ID NO: 1 (‘ClfA₄₀₋₅₅₉’). Thisfragments omits the long repetitive region towards the C-terminal of SEQID NO: 1.

clfB

The ‘clfB’ antigen is annotated as ‘clumping factor B’. In the NCTC 8325strain clfB is SAOUHSC_(—)02963 and has amino acid sequence SEQ ID NO: 3(GI:88196585). In the Newman strain it is nwmn_(—)2529 (GI:151222741).

Useful clfB antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 3 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 3; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 3, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These clfB proteins include variants of SEQ ID NO: 3.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 3. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 3 while retaining at least one epitopeof SEQ ID NO: 3. The final 40 C-terminal amino acids of SEQ ID NO: 3 canusefully be omitted. The first 44 N-terminal amino acids of SEQ ID NO: 3can usefully be omitted. Other fragments omit one or more proteindomains. ClfB is naturally a long protein and so the use of fragments ishelpful e.g. for purification, handling, fusion, expression, etc.

SEQ ID NO: 4 is a useful fragment of SEQ ID NO: 3 (‘ClfB₄₅₋₅₅₂’). Thisfragment includes the most exposed domain of ClfB and is more easilyused at an industrial scale. It also reduces the antigen's similaritywith human proteins. Other useful fragments, based on a 3-domain modelof ClfB, include: ClfB₄₅₋₃₆₀ (also known as CLfB-N12; SEQ ID NO: 5);ClfB₂₁₂₋₅₄₂ (also known as CLfB-N23; SEQ ID NO: 6); and ClfB₃₆₀₋₅₄₂(also known as CLfB-N3; SEQ ID NO: 7).

sdrE2

The ‘sdrE2’ antigen is annotated as ‘Ser-Asp rich fibrinogen/bonesialoprotein-binding protein SdrE’. In the Newman strain sdrE2 isNWMN_(—)0525 and has amino acid sequence SEQ ID NO: 8 (GI:151220737).

Useful sdrE2 antigens can elicit an antibody (e.g. when administered toa human) that recognises SEQ ID NO: 8 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 8; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 8, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These sdrE2 proteins include variants of SEQ ID NO:8. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 8.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 8 while retaining at least oneepitope of SEQ ID NO: 8. The final 38 C-terminal amino acids of SEQ IDNO: 8 can usefully be omitted. The first 52 N-terminal amino acids ofSEQ ID NO: 8 can usefully be omitted. Other fragments omit one or moreprotein domains. SdrE2 is naturally a long protein and so the use offragments is very helpful e.g. for purification, handling, fusion,expression, etc.

SEQ ID NO: 9 is a useful fragment of SEQ ID NO: 8 (‘SdrE₅₃₋₆₃₂’). Thisfragment includes the most exposed domain of SdrE2 and is more easilyused at an industrial scale. It also reduces the antigen's similaritywith human proteins.

sdrC

The ‘sdrC’ antigen is annotated as ‘sdrC protein’. In the NCTC 8325strain sdrC is SAOUHSC_(—)00544 and has amino acid sequence SEQ ID NO:10 (GI:88194324).

Useful sdrC antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 10 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 10; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 10, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These sdrC proteins include variants of SEQ ID NO:10. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 10.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 10 while retaining at least oneepitope of SEQ ID NO: 10. The final 38 C-terminal amino acids of SEQ IDNO: 10 can usefully be omitted. The first 50 N-terminal amino acids ofSEQ ID NO: 10 can usefully be omitted. Other fragments omit one or moreprotein domains. SdrC is naturally a long protein and so the use offragments is helpful e.g. for purification, handling, fusion,expression, etc.

SEQ ID NO: 11 is a useful fragment of SEQ ID NO: 10 (‘SdrC5₁₋₅₁₈’). Thisfragment includes the most exposed domain of SdrC and is more easilyused at an industrial scale. It also reduces the antigen's similaritywith human proteins.

sasF

The ‘sasF’ antigen is annotated as ‘sasF protein’. In the NCTC 8325strain sasF is SAOUHSC_(—)02982 and has amino acid sequence SEQ ID NO:12 (GI:88196601).

Useful sasF antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 12 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 12; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 12, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These sasF proteins include variants of SEQ ID NO:12. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 12.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 12 while retaining at least oneepitope of SEQ ID NO: 12. The final 39 C-terminal amino acids of SEQ IDNO: 12 can usefully be omitted. The first 37 N-terminal amino acids ofSEQ ID NO: 12 can usefully be omitted. Other fragments omit one or moreprotein domains.

emp

The ‘emp’ antigen is annotated as ‘extracellular matrix and plasmabinding protein’. In the NCTC 8325 strain emp is SAOUHSC_(—)00816 andhas amino acid sequence SEQ ID NO: 13 (GI:88194575). In the Newmanstrain it is nwmn_(—)0758 (GI:151220970).

Useful emp antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 13 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 13; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 13, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These emp proteins include variants of SEQ ID NO: 13.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 13. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 13 while retaining at least oneepitope of SEQ ID NO: 13. The first 26 N-terminal amino acids of SEQ IDNO: 13 can usefully be omitted. Other fragments omit one or more proteindomains.

SEQ ID NOs: 14, 15, 16 and 17 are useful fragments of SEQ ID NO: 13(‘Emp₃₅₋₃₄₀’, ‘Emp₂₇₋₃₃₄’, ‘Emp₃₅₋₃₃₄’ and ‘Emp₂₇₋₁₄₇’, respectively).

sdrD

The ‘sdrD’ antigen is annotated as ‘sdrD protein’. In the NCTC 8325strain sdrD is SAOUHSC_(—)00545 and has amino acid sequence SEQ ID NO:18 (GI:88194325).

Useful sdrD antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 18 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 18; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 18, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These sdrD proteins include variants of SEQ ID NO:18. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 18.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 18 while retaining at least oneepitope of SEQ ID NO: 18. The final 38 C-terminal amino acids of SEQ IDNO: 18 can usefully be omitted. The first 52 N-terminal amino acids ofSEQ ID NO: 18 can usefully be omitted. Other fragments omit one or moreprotein domains. SdrD is naturally a long protein and so the use offragments is very helpful e.g. for purification, handling, fusion,expression, etc.

SEQ ID NO: 19 is a useful fragment of SEQ ID NO: 18 (‘SdrD₅₃₋₅₉₂’). Thisfragment includes the most exposed domain of SdrD and is more easilyused at an industrial scale. It also reduces the antigen's similaritywith human proteins. Another useful fragment, with the same C-terminusresidue, is SdrD₃₉₄₋₅₉₂ (also known as SdrD-N3; SEQ ID NO: 20).

spa

The ‘spa’ antigen is annotated as ‘protein A’ or ‘SpA’. In the NCTC 8325strain spa is SAOUHSC_(—)00069 and has amino acid sequence SEQ ID NO: 21(GI:88193885). In the Newman strain it is nwmn_(—)0055 (GI:151220267).All S. aureus strains express the structural gene for spa, a wellcharacterized virulence factor whose cell wall-anchored surface proteinproduct has five highly homologous immunoglobulin binding domainsdesignated E, D, A, B, and C [230]. These domains display ˜80% identityat the amino acid level, are 56 to 61 residues in length, and areorganized as tandem repeats [231]. SpA is synthesized as a precursorprotein with an N-terminal signal peptide and a C-terminal sortingsignal [232,233]. Cell wall-anchored spa is displayed in great abundanceon the staphylococcal surface [234,235]. Each of its immunoglobulinbinding domains is composed of anti-parallel α-helices that assembleinto a three helix bundle and can bind the Fc domain of immunoglobulin G(IgG) [236,237], the VH3 heavy chain (Fab) of IgM (i.e. the B cellreceptor) [238], the von Willebrand factor at its A1 domain [239] and/orthe TNF-α receptor I (TNFRI) [240], which is displayed on surfaces ofairway epithelia.

Useful spa antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 21 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 21; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 21, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These spa proteins include variants of SEQ ID NO: 21.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 21. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 21 while retaining at least oneepitope of SEQ ID NO: 21. The final 35 C-terminal amino acids of SEQ IDNO: 21 can usefully be omitted. The first 36 N-terminal amino acids ofSEQ ID NO: 21 can usefully be omitted. Other fragments omit one or moreprotein domains. Reference 241 suggests that individual IgG-bindingdomains might be useful immunogens, alone or in combination.

SEQ ID NO: 22 is a useful fragment of SEQ ID NO: 21 (‘Spa₃₇₋₃₂₅’). Thisfragment contains all the five SpA Ig-binding domains and includes themost exposed domain of SpA. It also reduces the antigen's similaritywith human proteins. Other useful fragments may omit 1, 2, 3 or 4 of thenatural A, B, C, D and/or E domains. As reported in reference 241, otheruseful fragments may include only 1, 2, 3 or 4 of the natural A, B, C, Dand/or E domains e.g. comprise only the SpA(A) domain but not B to E, orcomprise only the SpA(D) domain but not A, B, C or E, etc. Thus a spaantigen useful with the invention may include 1, 2, 3, 4 or 5IgG-binding domains, but ideally has 4 or fewer. If an antigen includesonly one type of spa domain (e.g. only the Spa(A) or SpA(D) domain), itmay include more than one copy of this domain e.g. multiple SpA(D)domains in a single polypeptide chain. An individual domain within theantigen may be mutated at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aminoacids relative to SEQ ID NO: 21 (e.g. see ref. 241, disclosing mutationsat residues 3 and/or 24 of domain D, at residue 46 and/or 53 of domainA, etc.). Such mutants should not remove the antigen's ability to elicitan antibody that recognises SEQ ID NO: 21, but may remove the antigen'sbinding to IgG. In certain aspects a spa antigen includes a substitutionat (a) one or more amino acid substitution in an IgG Fc bindingsub-domain of SpA domain A, B, C, D and/or E that disrupts or decreasesbinding to IgG Fc, and (b) one or more amino acid substitution in aV_(H)3 binding sub-domain of SpA domain A, B, C, D, and/or E thatdisrupts or decreases binding to V_(H)3. In certain embodiments, avariant SpA comprises at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more variant SpA domain D peptides.

esaC

The ‘esaC’ antigen is annotated as ‘esaC’. In the NCTC 8325 strain esaCis SAOUHSC_(—)00264 and has amino acid sequence SEQ ID NO: 23(GI:88194069).

Useful esaC antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 23 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 23; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 23, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ormore). These esaC proteins include variants of SEQ ID NO: 23. Preferredfragments of (b) comprise an epitope from SEQ ID NO: 23. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or one or more aminoacids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from theN-terminus of SEQ ID NO: 23 while retaining at least one epitope of SEQID NO: 23. Other fragments omit one or more protein domains.

esxA

The ‘esxA’ antigen is annotated as ‘protein’. In the NCTC 8325 strainesxA is SAOUHSC_(—)00257 and has amino acid sequence SEQ ID NO: 24(GI:88194063).

Useful esxA antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 24 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 24; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 24, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more).These esxA proteins include variants of SEQ ID NO: 24. Preferredfragments of (b) comprise an epitope from SEQ ID NO: 24. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or one or more aminoacids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from theN-terminus of SEQ ID NO: 24 while retaining at least one epitope of SEQID NO: 24. Other fragments omit one or more protein domains.

esxB

The ‘esxB’ antigen is annotated as ‘esxB’. In the NCTC 8325 strain esxBis SAOUHSC_(—)00265 and has amino acid sequence SEQ ID NO: 25(GI:88194070).

Useful esxB antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 25 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 25; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 25, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ormore). These esxB proteins include variants of SEQ ID NO: 25. Preferredfragments of (b) comprise an epitope from SEQ ID NO: 25. Other preferredfragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25 or more) from the C-terminus and/or one or more aminoacids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from theN-terminus of SEQ ID NO: 25 while retaining at least one epitope of SEQID NO: 25. Other fragments omit one or more protein domains.

sta006

The ‘sta006’ antigen is annotated as ‘ferrichrome-binding protein’, andhas also been referred to as ‘FhuD2’ in the literature [242]. In theNCTC 8325 strain sta006 is SAOUHSC_(—)02554 and has amino acid sequenceSEQ ID NO: 26 (GI:88196199). In the Newman strain it is nwmn_(—)2185(GI:151222397).

Useful sta006 antigens can elicit an antibody (e.g. when administered toa human) that recognises SEQ ID NO: 26 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 26; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 26, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These sta006 proteins include variants of SEQ ID NO:26. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 26.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 26 while retaining at least oneepitope of SEQ ID NO: 26. The first 17 N-terminal amino acids of SEQ IDNO: 26 can usefully be omitted. Other fragments omit one or more proteindomains. Mutant forms of sta006 are reported in reference 243. A sta006antigen may be lipidated e.g. with an acylated N-terminus cysteine.

isdC

The ‘isdC’ antigen is annotated as ‘protein’. In the NCTC 8325 strainisdC is SAOUHSC_(—)01082 and has amino acid sequence SEQ ID NO: 27(GI:88194830).

Useful isdC antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 27 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 27; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 27, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200 or more). These isdC proteins include variants of SEQ ID NO: 27.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 27. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 27 while retaining at least oneepitope of SEQ ID NO: 27. The final 39 C-terminal amino acids of SEQ IDNO: 27 can usefully be omitted. The first 28 N-terminal amino acids ofSEQ ID NO: 27 can usefully be omitted. Other fragments omit one or moreprotein domains. Useful fragments of IsdB are disclosed in reference249.

Reference 244 discloses antigens which usefully include epitopes fromboth IsdB and IsdH.

Hla

The ‘Hla’ antigen is the ‘alpha-hemolysin precursor’ also known as‘alpha toxin’ or simply ‘hemolysin’. In the NCTC 8325 strain Hla isSAOUHSC_(—)01121 and has amino acid sequence SEQ ID NO: 28(GI:88194865). In the Newman strain it is nwmn_(—)1073 (GI:151221285).Hla is an important virulence determinant produced by most strains of S.aureus, having pore-forming and haemolytic activity. Anti-Hla antibodiescan neutralise the detrimental effects of the toxin in animal models,and Hla is particularly useful for protecting against pneumonia.

Useful Hla antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 28 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 28; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 28, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These Hla proteins include variants of SEQ ID NO: 28.Preferred fragments of (b) comprise an epitope from SEQ ID NO: 28. Otherpreferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or moreamino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more)from the N-terminus of SEQ ID NO: 28 while retaining at least oneepitope of SEQ ID NO: 28. The first 26 N-terminal amino acids of SEQ IDNO: 28 can usefully be omitted. Truncation at the C-terminus can also beused e.g. leaving only 50 amino acids (residues 27-76 of SEQ ID NO: 28)[245]. Other fragments omit one or more protein domains.

Hla's toxicity can be avoided in compositions of the invention bychemical inactivation (e.g. using formaldehyde, glutaraldehyde or othercross-linking reagents). Instead, however, it is preferred to use mutantforms of Hla which remove its toxic activity while retaining itsimmunogenicity. Such detoxified mutants are already known in the art.One useful Hla antigen has a mutation at residue 61 of SEQ ID NO: 28,which is residue 35 of the mature antigen (i.e. after omitting the first26 N-terminal amino acids). Thus residue 61 may not be histidine, andmay instead be e.g. Ile, Val or preferably Leu. A His-Arg mutation atthis position can also be used. For example, SEQ ID NO: 29 is the maturemutant Hla-H35L sequence and a useful Hla antigen comprises SEQ ID NO:29. Another useful mutation replaces a long loop with a short sequencee.g. to replace the 39mer at residues 136-174 of SEQ ID NO: 28 with atetramer such as PSGS (SEQ ID NO: 30), as in SEQ ID NO: 31 (which alsoincludes the H35L mutation) and SEQ ID NO: 32 (which does not includethe H35L mutation).

Further useful Hla antigens are disclosed in references 246 and 247.

SEQ ID NOs: 33, 34 & 35 are three useful fragments of SEQ ID NO: 28(‘Hla₂₇₋₇₆’, ‘Hla₂₇₋₈₉’ and ‘Hla₂₇₋₇₉’, respectively). SEQ ID NOs: 36,37 and 38 are the corresponding fragments from SEQ ID NO: 29.

sta011

The ‘sta011’ antigen is annotated as ‘lipoprotein’. In the NCTC 8325strain sta011 is SAOUHSC_(—)00052 and has amino acid sequence SEQ ID NO:39 (GI:88193872).

Useful sta011 antigens can elicit an antibody (e.g. when administered toa human) that recognises SEQ ID NO: 39 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 39; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 39, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These sta011 proteins include variants of SEQ ID NO:39. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 39.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 39 while retaining at least oneepitope of SEQ ID NO: 39. The first 23 N-terminal amino acids of SEQ IDNO: 39 can usefully be omitted. Other fragments omit one or more proteindomains. A sta006 antigen may be lipidated e.g. with an acylatedN-terminus cysteine.

Variant forms of SEQ ID NO: 39 which may be used for preparing sta011antigens include, but are not limited to, SEQ ID NOs: 40, 41 and 42 withvarious Ile/Val/Leu substitutions.

isdA

The ‘isdA’ antigen is annotated as ‘IsdA protein’. In the NCTC 8325strain isdA is SAOUHSC_(—)01081 and has amino acid sequence SEQ ID NO:43 (GI:88194829). In the Newman strain it is nwmn_(—)1041(GI:151221253).

Useful isdA antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 43 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 43; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 43, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These isdA proteins include variants of SEQ ID NO:43. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 43.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 43 while retaining at least oneepitope of SEQ ID NO: 43. The final 38 C-terminal amino acids of SEQ IDNO: 43 can usefully be omitted. The first 46 N-terminal amino acids ofSEQ ID NO: 43 can usefully be omitted. Truncation to exclude theC-terminal 38mer of SEQ ID NO: 43 (beginning with the LPKTG motif) isalso useful. Other fragments omit one or more protein domains.

SEQ ID NO: 44 is a useful fragment of SEQ ID NO: 43 (amino acids 40-184of SEQ ID NO: 43; ‘IsdA₄₀₋₁₈₄’) which includes the natural protein'sheme binding site and includes the antigen's most exposed domain. Italso reduces the antigen's similarity with human proteins. Other usefulfragments are disclosed in references 248 and 249.

IsdA does not adsorb well to aluminium hydroxide adjuvants, so IsdApresent in a composition may me unadsorbed or may be adsorbed to analternative adjuvant e.g. to an aluminium phosphate.

isdB

The ‘isdB’ antigen is annotated as ‘neurofilament protein isdB’. In theNCTC 8325 strain isdB is SAOUHSC_(—)01079 and has amino acid sequenceSEQ ID NO: 45 (GI:88194828). IsdB has been proposed for use as a vaccineantigen on its own [250], but this may not prevent pneumonia.

Useful isdB antigens can elicit an antibody (e.g. when administered to ahuman) that recognises SEQ ID NO: 45 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 45; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 45, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These isdB proteins include variants of SEQ ID NO:45. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 45.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 45 while retaining at least oneepitope of SEQ ID NO: 45. The final 36 C-terminal amino acids of SEQ IDNO: 45 can usefully be omitted. The first 40 N-terminal amino acids ofSEQ ID NO: 45 can usefully be omitted. Other fragments omit one or moreprotein domains. Useful fragments of IsdB are disclosed in references249 and 251 e.g. lacking 37 internal amino acids of SEQ ID NO: 45.

In some embodiments, compositions of the invention do not include anisdB antigen.

sta073

The ‘sta073’ antigen is annotated as ‘bifunctional autolysin precursor’.In the NCTC 8325 strain sta073 is SAOUHSC_(—)00994 and has amino acidsequence SEQ ID NO: 46 (GI:88194750). In the Newman strain it isnwmn_(—)0922 (GI:151221134). Proteomic analysis has revealed that thisprotein is secreted or surface-exposed.

Useful sta073 antigens can elicit an antibody (e.g. when administered toa human) that recognises SEQ ID NO: 46 and/or may comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 46; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 46, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). These sta073 proteins include variants of SEQ ID NO:46. Preferred fragments of (b) comprise an epitope from SEQ ID NO: 46.Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one ormore amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 ormore) from the N-terminus of SEQ ID NO: 46 while retaining at least oneepitope of SEQ ID NO: 46. The first 24 N-terminal amino acids of SEQ IDNO: 46 can usefully be omitted. Other fragments omit one or more proteindomains.

Sta073 does not adsorb well to aluminium hydroxide adjuvants, so Sta073present in a composition may me unadsorbed or may be adsorbed to analternative adjuvant e.g. to an aluminium phosphate.

Hybrid Polypeptides

S. aureus protein antigens used in the invention may be present in thecomposition as individual separate polypeptides. Where more than oneantigen is used, however, they do not have to be present as separatepolypeptides. Instead, at least two (e.g. 2, 3, 4, 5, or more) antigenscan be expressed as a single polypeptide chain (a ‘hybrid’ polypeptide).Hybrid polypeptides offer two main advantages: first, a polypeptide thatmay be unstable or poorly expressed on its own can be assisted by addinga suitable hybrid partner that overcomes the problem; second, commercialmanufacture is simplified as only one expression and purification needbe employed in order to produce two polypeptides which are bothantigenically useful.

The hybrid polypeptide may comprise two or more polypeptide sequencesfrom each of the antigens listed above, or two or more variants of thesame antigen in the cases in which the sequence has partial variabilityacross strains.

Hybrids consisting of amino acid sequences from two, three, four, five,six, seven, eight, nine, or ten antigens are useful. In particular,hybrids consisting of amino acid sequences from two, three, four, orfive antigens are preferred, such as two or three antigens.

Different hybrid polypeptides may be mixed together in a singleformulation. Hybrids may be combined with non-hybrid antigens selectedfrom the first, second or third antigen groups. Within suchcombinations, an antigen may be present in more than one hybridpolypeptide and/or as a non-hybrid polypeptide. It is preferred,however, that an antigen is present either as a hybrid or as anon-hybrid, but not as both.

Hybrid polypeptides can be represented by the formulaNH₂-A-{-X-L-}_(n)-B—COOH, wherein: X is an amino acid sequence of a S.aureus antigen, as described above; L is an optional linker amino acidsequence; A is an optional N-terminal amino acid sequence; B is anoptional C-terminal amino acid sequence; n is an integer of 2 or more(e.g. 2, 3, 4, 5, 6, etc.). Usually n is 2 or 3.

If a —X— moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the hybrid protein. In someembodiments, the leader peptides will be deleted except for that of the—X— moiety located at the N-terminus of the hybrid protein i.e. theleader peptide of X₁ will be retained, but the leader peptides of X₂ . .. X_(n) will be omitted. This is equivalent to deleting all leaderpeptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of {-X-L-}, linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOH,NH₂—X₁—X₂-L₂-COOH, etc. Linker amino acid sequence(s) -L- will typicallybe short (e.g. 20 or fewer amino acids i.e. 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise shortpeptide sequences which facilitate cloning, poly-glycine linkers (i.e.comprising Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), andhistidine tags (i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more).Other suitable linker amino acid sequences will be apparent to thoseskilled in the art. A useful linker is GSGGGG (SEQ ID NO: 47) orGSGSGGGG (SEQ ID NO: 48), with the Gly-Ser dipeptide being formed from aBamH1 restriction site, thus aiding cloning and manipulation, and the(Gly)₄ tetrapeptide being a typical poly-glycine linker. Other suitablelinkers, particularly for use as the final L_(n) are ASGGGS (SEQ ID NO:49 e.g. encoded by SEQ ID NO: 50) or a Leu-Glu dipeptide.

-A- is an optional N-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 40, 39, 38, 37, 36, 35, 34,33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includeleader sequences to direct protein trafficking, or short peptidesequences which facilitate cloning or purification (e.g. histidine tagsi.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A- is preferably anoligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) whichprovides a N-terminus methionine e.g. Met-Ala-Ser, or a single Metresidue.

—B— is an optional C-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g. comprising histidine tags i.e.His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more, such as SEQ ID NO: 51),or sequences which enhance protein stability. Other suitable C-terminalamino acid sequences will be apparent to those skilled in the art.

One hybrid polypeptide of the invention may include both EsxA and EsxBantigens. These may be in either order, N- to C-terminus. SEQ ED NOs: 52(‘EsxAB’; encoded by SEQ ID NO: 53) and 54 (‘EsxBA’) are examples ofsuch hybrids, both having hexapeptide linkers ASGGGS (SEQ ID NO: 49).

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references252-259, etc.

“GI” numbering is used above. A GI number, or “GenInfo Identifier”, is aseries of digits assigned consecutively to each sequence recordprocessed by NCBI when sequences are added to its databases. The GInumber bears no resemblance to the accession number of the sequencerecord. When a sequence is updated (e.g. for correction, or to add moreannotation or information) then it receives a new GI number. Thus thesequence associated with a given GI number is never changed.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.260. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. 261.

Where the invention concerns an “epitope”, this epitope may be a B-cellepitope and/or a T-cell epitope. Such epitopes can be identifiedempirically (e.g. using PEPSCAN [262,263] or similar methods), or theycan be predicted (e.g. using the Jameson-Wolf antigenic index [264],matrix-based approaches [265], MAPITOPE [266], TEPITOPE [267,268],neural networks [269], OptiMer & EpiMer [270, 271], ADEPT [272], Tsites[273], hydrophilicity [274], antigenic index [275] or the methodsdisclosed in references 276-280, etc.). Epitopes are the parts of anantigen that are recognised by and bind to the antigen binding sites ofantibodies or T-cell receptors, and they may also be referred to as“antigenic determinants”.

Where an antigen “domain” is omitted, this may involve omission of asignal peptide, of a cytoplasmic domain, of a transmembrane domain, ofan extracellular domain, etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Where the invention provides a process involving multiple sequentialsteps, the invention can also provide a process involving less than thetotal number of steps. For example, the invention provides a processcomprising the steps of: (a) depolymerising a S. aureus type 5 or type 8capsular polysaccharide, to give a polysaccharide fragment; and (b)oxidising the fragment in order to introduce an aldehyde group into atleast one saccharide residue in the fragment, to give an oxidisedsaccharide residue. The further step (c) need not be performed in orderto fall within the scope of the invention, as the product of steps (a)and (b) has utility as an intermediate in conjugate preparation, and maybe used, stored, exported, etc. for separate and later use. This lateruse might involve carrying out step (c). Alternatively, the product ofsteps (a) and (b) might be coupled to a carrier molecule in a differentway, for example via a hydroxyl or carboxyl group that is retained inthe oxidised saccharide residue.

Similarly, where a starting polysaccharide material is already partiallyprocessed then the invention encompasses processes involving only thelater steps of a method. For example, the invention encompasses aprocess comprising a step of coupling an oxidised saccharide residue ina type 5 or type 8 capsular polysaccharide to a carrier molecule via analdehyde group, in which the starting material for the process is a type5 or type 8 capsular polysaccharide that was previously depolymerised togive a polysaccharide fragment and then oxidised to introduce an thealdehyde group into the saccharide residue.

These different steps can be performed at very different times bydifferent people in different places (e.g. in different countries).

It will be appreciated that sugar rings can exist in open and closedform and that, whilst closed forms are shown in structural formulaeherein, open forms are also encompassed by the invention. Similarly, itwill be appreciated that sugars can exist in pyranose and furanose formsand that, whilst pyranose forms are shown in structural formulae herein,furanose forms are also encompassed. Different anomeric forms of sugarsare also encompassed.

A primary amine can be represented by formula NH₂R. The R group willtypically be electron donating, and includes C₁₋₈hydrocarbyl,particularly C₁₋₈alkyl, especially methyl. R is often —CH₃, —C₂H₅ or—C₃H₇. The hydrocarbyl may be substituted with one or more groups, suchas: halogen (e.g. Cl, Br, F, I), trihalomethyl, —NO₂, —CN,—N⁺(C₁₋₆alkyl)₂O⁻, —SO₃H, —SOC₁₋₆alkyl, —SO₂C₁₋₆alkyl, —SO₃C₁₋₆alkyl,—OC(═O)OC₁₋₆alkyl, —C(═O)H, —C(═O)C₁₋₆alkyl, —OC(═O)C₁₋₆alkyl,—N(C₁₋₆alkyl)₂, C₁₋₆alkyl, —N(C₁₋₆alkyl)₂, —C(═O)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)C(═O)O(C₁₋₆alkyl), —N(C₁₋₆alkyl)C(═O)N(C₁₋₆ alkyl)₂, —CO₂H,—OC(═O)N(C₁₋₆alkyl)₂, —N(C₁₋₆alkyl)C(═O)C₁₋₆alkyl,—N(C₁₋₆alkyl)C(═S)C₁₋₆alkyl, —N(C₁₋₆alkyl)SO₂N(C₁₋₆alkyl)₂,—CO₂C₁₋₆alkyl, —SO₂N(C₁₋₆alkyl)₂, —C(═O)NH₂, —C(═S)N(C₁₋₆alkyl)₂,—N(C₁₋₆alkyl)SO₂C₁₋₆alkyl, —N(C₁₋₆ alkyl)C(═S)N(C₁₋₆alkyl)₂,—NH—C₁₋₆alkyl, —S—C₁₋₆alkyl or —O—C₁₋₆alkyl. The term ‘hydrocarbyl’includes linear, branched or cyclic monovalent groups consisting ofcarbon and hydrogen. Hydrocarbyl groups thus include alkyl, alkenyl andalkynyl groups, cycloalkyl (including polycycloalkyl), cycloalkenyl andaryl groups and combinations thereof, e.g. alkylcycloalkyl,alkylpolycycloalkyl, alkylaryl, alkenylaryl, cycloalkylaryl,cycloalkenylaryl, cycloalkylalkyl, polycycloalkylalkyl, arylalkyl,arylalkenyl, arylcycloalkyl and arylcycloalkenyl groups. Typicalhydrocarbyl are C₁₋₁₄ hydrocarbyl, more particularly C₁₋₈ hydrocarbyl.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a scheme for making an S. aureus type 5 capsularpolysaccharide-CRM197 conjugate using an adipic acid dihydrazine linkerand carbodiimide chemistry.

FIG. 2 a shows an SDS-PAGE analysis of the S. aureus type 5 capsularpolysaccharide-CRM197 conjugate made using an adipic acid dihydrazinelinker and carbodiimide chemistry. FIG. 2 b shows an S300 SEPHACRYL™chromatogram of the S. aureus type 5 capsular polysaccharide-CRM197conjugate made using an adipic acid dihydrazine linker and carbodiimidechemistry.

FIG. 3 shows an S300 SEPHACRYL™ chromatogram of depolymerised type 5capsular polysaccharide.

FIG. 4A compares 1D ¹H signals in the ¹H anomeric region fordepolymerised and native type 5 capsular polysaccharide. Some notabledifferences are marked. FIGS. 4B and 4C compare the anomeric andMethyl-Fucose regions respectively of 2D (¹H, ¹H) scalar couplingspectra for these polysaccharides. Some notable differences are marked.

FIG. 5 a shows an SDS-PAGE analysis of a S. aureus type 5 capsularpolysaccharide-CRM197 conjugate made using a method of the invention.FIG. 5 b shows an S300 SEPHACRYL™ chromatogram of a S. aureus type 5capsular polysaccharide-CRM197 conjugate made using a method of theinvention.

FIG. 6 shows an S300 SEPHACRYL™ chromatogram of S. aureus type 5capsular polysaccharides depolymerised under various conditions.

FIG. 7 shows SDS-PAGE analyses of S. aureus type 5 capsularpolysaccharide-CRM197 conjugates made using methods of the invention.

FIG. 8 shows the IgG response to various antigens in a mouse kidneyabscess model of S. aureus infection.

FIG. 9 compares IgG and IgM responses to various antigens in the mousekidney abscess model.

FIG. 10 shows protective responses to various antigens in the mousekidney abscess model.

FIG. 11 compares the IgG response to different conjugates in the mousekidney abscess model.

FIG. 12 compares protective responses to different conjugates in themouse kidney abscess model.

FIGS. 13A and 13B compare protective responses to further conjugates inthe mouse kidney abscess model.

FIG. 14 compares IgG and IgM responses to further conjugates in themouse kidney abscess model.

FIG. 15 compares protective responses to a further conjugate whenadjuvanted with different agents in the mouse kidney abscess model.

FIG. 16 compares responses to various antigens in a mouse lethal modelof S. aureus infection.

FIG. 17 shows a SEC-HPLC chromatogram of S. aureus type 8 capsularpolysaccharide depolymerised with 2M hydrochloric acid.

FIG. 18 shows an NMR spectrum of S. aureus type 8 capsularpolysaccharide depolymerised with 2M hydrochloric acid.

MODES FOR CARRYING OUT THE INVENTION Conjugate Production andCharacterisation

A purified S. aureus type 5 capsular polysaccharide was conjugated toCRM197 using carbodiimide chemistry and an adipic acid dihydrazinelinker, similar to the method used in reference 2 (see below). In thismethod, the capsular polysaccharide is conjugated to derivatised CRM197using EDC (FIG. 1). The reaction involves the carboxyl groups of thecapsular polysaccharide. The carbodiimide (EDC) activates the carboxylgroups to bind to the —NH₂ group from the derivatised carrier protein(CRMadh), forming an amide linkage. The derivatised CRMadh is preparedusing the same carbodiimide chemistry.

CRMadh Preparation:

To a solution of CRM197 was added 100 mM MES pH6.0 buffer in order toreach a final concentration of 10-12 mg/ml. Then 3.5 mg/ml of ADH(adipic acid dihydrazide) and 0.15 (EDC/CRM, w/w) was added, and thereaction kept under mild stirring for 1 h at RT. The mixture was thendialyzed against first 200 mM NaCl, 10 mM MES pH7.3 buffer and thenagainst 5 mM MES pH7.0 buffer, using a 6-8 kDa membrane (SpectraPor).The product was characterized by MicroBCA, SDS-Page (3-8%), HPLC and MS.The CRMadh was found to be derivatised with 6-8 linker of ADH).

Conjugation Reaction:

The conjugation reaction was performed at capsular polysaccharideconcentration of 2 mg/mL in 50 mM MES buffer pH6.04. The derivatisedcarrier protein, CRMadh, was added to the solution of capsularpolysaccharide to a final concentration of 4.0 mg/ml. The solution waskept at RT for 3 h. The polysaccharide:protein ratio in the reactionmixture was 1:2 (weight/weight), the polysaccharide:EDC ratio was 1:6.66(equivalent/equivalent) and the polysaccharide:SulfoNHS ratio was 1:0.53(equivalent/equivalent).

After 3 h, formation of the conjugate was verified by SDS-PAGE using aNuPAGE® 3-8% Tris-Acetate Gel (Invitrogen) (FIG. 2 a). Afterconjugation, the conjugate was purified by gel-filtration chromatography(performed on an AKTA™ system (G&E Healthcare) using a S300 SEPHACRYL™resin (G&E Healthcare), with a 10 mM NaPi, 10 mM NaCl, pH7.2 mobilephase buffer). The conjugate was detected at 215 nm, 254 nm and 280 nm(FIG. 2 b). The conjugate solution was stored at −20° C. until furtheruse. Total saccharide in the conjugate was determined by HPAEC-PADanalysis and protein content by MicroBCA assay, as described inreference 281 (Table 1).

TABLE 1 Conjugate Protein Saccharide Saccharide/ (lot) (μg/ml) (μg/ml)protein (w/w) 1 26.00 12.10 0.47 2 33.90 11.00 0.32 3 62.21 29.40 0.47 445.21 9.30 0.21

Purified S. aureus type 5 and type 8 capsular polysaccharides wereseparately conjugated to CRM197 using a method of the invention (seebelow).

Depolymerisation

Purified capsular polysaccharide was dissolved in distilled water at 2mg/mL. Acetic acid was added to a final concentration of 2% (v/v) andthe reaction kept at 90° C. for 3 hours (or overnight in the case of LotB). The solution was then neutralized with 1M NaOH and the depolymerisedpolysaccharide purified on a gel-filtration column (performed on anAKTA™ system (G&E Healthcare) using a S300 SEPHACRYL™ resin (G&EHealthcare), with a 10 mM NaPi, 10 mM NaCl, pH7.2 mobile phase buffer).The saccharide was detected at 215 nm (FIG. 3). Pooled fractions weredialyzed against distilled water using a 1 kDa membrane (SpectraPor) andlyophilized.

The site of cleavage was verified as being at (1→3) glycosidic linkageswithin the type 5 polysaccharide using ¹H NMR. Briefly, samples ofnative and depolymerised type 5 capsular polysaccharide werefreeze-dried to eliminate protonated water solvent and dissolved indeuterium oxide (99.9% deuterium, Sigma-Aldrich). All NMR spectra wererecoded at 50° C. on a Bruker Avance III 400 MHz spectrometer using a5-mm broadband probe and the TopSpin 2.1 software package (Bruker) fordata acquisition and processing. ID ¹H spectra were collected using astandard one-pulse experiment over a spectral width of 4,000 Hz andcollecting 32k data points. The transmitter was set at the residual HDOfrequency (4.79 ppm). The spectra were obtained in a quantitative mannerusing a total recycle time to ensure a full recovering of each signal(5× Longitudinal Relaxation Time T1). Spectra were Fourier transformedafter applying a 0.2 Hz line broadening function. 2D (¹H, ¹H) scalarcorrelation spectra were recorded by DQF-COSY pulse sequence. 4096 datapoints were collected in the F2 domain and 256 in the F1 domain.

ID ¹H signals for the native polysaccharide were compared with publishedvalues and found to be in agreement (Table 2).

TABLE 2 Measured Signal δ (ppm)* Pubd. δ (ppm)** Pubd. δ (ppm)*** H₃^(L-FucNAc-OAc) 4.958 5.005 5.005 H₁ ^(L-FucNAc-OAc) 4.929 4.981 4.975H₃ ^(L-FucNAc-deOAc) 4.864 4.935 4.911 H₁ ^(ManNAc-deOAc) 4.801 4.8604.847 H₁ ^(ManNAc-OAc) 4.638 4.698 4.683 H₂ ^(ManNAc-deOAc) 4.625 4.6804.670 H₂ ^(ManNAc-deOAc) 4.584 4.645 4.629 H₁ ^(D-FucNAc-OAc/deOAc)4.405 4.461 4.452 H₄ ^(L-FucNAc-OAc) 4.320 4.382 4.367 H₂^(L-FucNAc-OAc) 4.292 4.368 4.338 H₅ ^(L-FucNAc-OAc) 4.121 4.175 4.168H₅ ^(L-FucNAc-deOAc) 4.080 4.142 4.126 H₂ ^(L-FucNAc-deOAc) 4.033 4.1054.077 H₄ ^(L-FucNAc-deOAc) 4.005 4.060 4.051 NAc^(D-FucNAc-OAC) 2.0832.149 2.131 NAc^(D-FucNAc-deOAc) 2.067 2.126 2.115 OAc^(L-FucNAc-OAc)2.004 2.070 2.051 NAc^(L-FucNAc-deOAc) 1.995 2..057 2.002NAc^(L-FucNAc-OAc) 1.955 2.023 2.043 NAc^(ManNAc-deOAc) 1.948 2.0181.996 NAc^(ManNAc-OAc) 1.943 2.011 1.992 H₆ ^(D-FucNAc-OAC) 1.238 1.3001.287 H₆ ^(D-FucNAc-deOAc) 1.298 H₆ ^(L-FucNAc-OAc/deOAc) 1.183 1.2421.231 *HDO signal at 4.484 ppm **Jones C. Carbohydr. Res. 2005, 340(6),1097-1106 - HDO signal at 4.484 ppm ***Jones C. Carbohydr. Res. 2005,340(6), 1097-1106 - H1L-FucNAc = 5.005 ppm, therefore HDO signal at4.532 ppm instead of 4.484 ppm

FIG. 4A compares 1D ¹H signals in the ¹H anomeric region for thedepolymerised and native polysaccharides. FIGS. 4B and 4C compare theanomeric and Methyl-Fucose regions respectively of 2D (¹H, ¹H) scalarcoupling spectra for these polysaccharides. The data show that theacetic acid treatment resulted in cleavage of (1→3) glycosidic linkagesbetween the α-L-FucNAc(3OAc) and β-D-FucNAc residues in the type 5capsular polysaccharide.

Oxidation

The depolymerised capsular polysaccharide was dissolved in distilledwater at 2 mg/mL. NaIO₄ was added at a polysaccharide:NaIO₄ ratio of 1:1(weight/weight) and the reaction kept at room temperature for 1-2 hoursin the dark. The solution was then dialyzed against distilled waterusing a 1 kDa membrane (SpectraPor) and lyophilized once again.

Conjugation

The oxidised capsular polysaccharide was dissolved in a 200 mM NaPi, 1MNaCl, pH7.2 buffer at a concentration of 10 mg/mL. CRM197 was added tothe solution at a polysaccharide:protein ratio of 4:1 (weight/weight)and NaBH₃CN (Aldrich) added at a sacchaaride:NaBCNH₃ ratio of 2:1(weight/weight). The solution was kept at 37° C. for 2 days. SDS-PAGEwas used to confirm formation of the conjugate (see FIG. 5 a for thetype 5 conjugate). After conjugation, the conjugate was purified bygel-filtration chromatography (performed on an AKTA™ system (G&EHealthcare) using a S300 SEPHACRYL™ resin (G&E Healthcare), with a 10 mMNaPi, 10 mM NaCl, pH7.2 mobile phase buffer). The conjugate was detectedat 215 nm, 254 nm and 280 nm (see FIG. 5 b for the type 5 conjugate).The conjugate solution was stored at −20° C. until further use. Totalsaccharide in the conjugate was determined by HPAEC-PAD analysis andprotein content by MicroBCA assay (see Table 3a for the type 5 conjugateand Table 3b for type 8 conjugate).

TABLE 3a Saccharide/ Conjugate (lot) Protein (μg/ml) Saccharide (μg/ml)protein (w/w) A 51.52 1.72 0.03 B 161.80 17.10 0.11 C 34.42 4.22 0.12 D40.56 12.70 0.31 E 196.00 55.17 0.28

TABLE 3b Saccharide/ Conjugate (lot) Protein (μg/ml) Saccharide (μg/ml)protein (w/w) α 518.00 82.30 0.16 β 11.00 7.94 0.72 γ 23.22 5.57 0.24 δ22.87 5.08 0.22

Purified S. aureus type 5 was conjugated to CRM197 using another methodof the invention. In this method, the depolymerisation, oxidation andconjugation steps were carried out as described above, except that theconjugation step was carried out with the derivatised carrier proteindescribed above (CRMadh) instead of CRM197. Total saccharide in theconjugate was determined by HPAEC-PAD analysis and protein content byMicroBCA assay (Table 4).

TABLE 4 Saccharide Conjugate (lot) Protein (μg/ml) (μg/ml)Saccharide/protein (w/w) A′ 58.25 2.49 0.043

Alternative Depolymerisation Methods

In other studies, different conditions were tested for depolymerisationof the purified capsular polysaccharide. The polysaccharide wasdissolved in distilled water at 2 mg/mL. Acetic acid was added to afinal concentration of 2% or 5% (v/v) and the reaction kept at 90° C.for 30 minutes, 3 hours, 5 hours or 6 hours. The solution was thenneutralized and purified on a gel-filtration column as described above.The saccharide was detected at 215 nm and pooled (FIG. 6).

The pooled fractions were then oxidised and dialyzed against water asdescribed above. The fractions were conjugated to CRM197 or CRMadh asdescribed above and the resultant conjugates purified by gel-filtrationchromatography also as described above (FIG. 7).

In another study, hydrochloric acid at 0.5M was used instead of aceticacid for type 8 capsular polysaccharide, and the reaction kept at 90° C.for 2.5 hours, with the reaction being sampled every 30 minutes. Sampleswere analysed by NMR and SEC-HPLC. No depolymerisation was observed, andthe level of O-acetylation remained almost unchanged. In contrast, whenhydrochloric acid at 2M was used, and the reaction kept at 100° C.,depolymerisation was observed even after only 30 minutes. The level ofO-acetylation gradually fell over the 2.5 hours (FIGS. 17 and 18 (withacetyl peak circled)).

Immunisation Study—Abscess Model (1)

General Assay Protocol:

Mice were immunized according to the schedule described below andchallenged by intravenous injection of a bacterial suspension of S.aureus. The culture of S. aureus was centrifuged, washed twice anddiluted in PBS before challenge. Further dilutions were needed for thedesired inoculum, which was experimentally verified by agar plating andcolony formation. For organ harvest, mice were euthanized and theirkidneys removed and homogenized in 1% TRITON™ X-100. Aliquots were thendiluted and plated on agar media for triplicate determination of CFU.For histology, kidney tissue was incubated at room temperature in 10%formalin for 24 hours. Tissues were embedded in paraffin, thinsectioned, hematoxylin/eosin stained and examined by microscopy.

CD1 mice at 3 weeks old were immunised at days 0 and 11 byintraperitoneal injection with a 5 μg dose of antigen in an injectionvolume of 200 μl. The mice were bled on days 0 and 20 and challengedwith S. aureus on day 21. Organs were harvested at day 25. Immunisationswere carried out in groups of eight mice according to the followingscheme:

-   -   Group 1—Alum alone    -   Group 2—Type 5 capsular polysaccharide alone    -   Group 3—Type 5 capsular polysaccharide plus alum    -   Group 4—Type 5 capsular polysaccharide-CRMadh conjugate (Lot 1)    -   Group 5—Type 5 capsular polysaccharide-CRMadh conjugate (Lot 1)        plus alum

The conjugate induced a specific IgG response against type 5polysaccharide. The alum formulation gave an improved response (FIG. 8).The conjugate also induced a specific IgM response against type 5polysaccharide (FIG. 9). The alum conjugate formulation also gave thebest protection from kidney infection (FIG. 10).

Immunisation Study—Abscess Model (2)

CD1 mice at 3 weeks old were immunised at days 1, 14 and 28 byintraperitoneal injection with a 5 μg dose of antigen in an injectionvolume of 200 μl. The mice were bled on days 0, 27 and 37 and challengedwith S. aureus on day 38. Organs were harvested at day 42. Immunisationswere carried out in groups of eight mice according to the followingscheme:

-   -   Group 1—Alum alone    -   Group 2—Type 5 capsular polysaccharide plus alum    -   Group 3—Type 5 capsular polysaccharide-CRMadh conjugate (Lot 2)        plus alum    -   Group 4—Type 5 capsular polysaccharide-CRMadh conjugate (Lot A′)        plus alum

The conjugates induced a specific IgG response against type 5polysaccharide. The conjugates of the invention (represented by lot A′)gave a particularly high titre (FIG. 11). The conjugates of theinvention gave the best protection from kidney infection (FIG. 12).

Immunisation Study—Abscess Model (3)

CD1 mice at 3 weeks old were immunised at days 1, 14 and 28 byintraperitoneal injection with a 5 μg dose (or 0.5 μg dose in the caseof lot A) of antigen in an injection volume of 200 μl. The mice werebled on days 0, 27 and 37 and challenged with S. aureus (grown in liquidor solid medium) on day 38. Organs were harvested at day 42.Immunisations were carried out in groups of eight mice according to thefollowing scheme:

-   -   Group 1—Alum alone    -   Group 2—Type 5 capsular polysaccharide plus alum    -   Group 3—Type 5 capsular polysaccharide-CRMadh conjugate (Lot 2)        plus alum    -   Group 4—Type 5 capsular polysaccharide-CRMadh conjugate (Lot 3)        plus alum    -   Group 5—Type 5 capsular polysaccharide-CRMadh conjugate (Lot A′)        plus alum    -   Group 6—Type 5 capsular polysaccharide-CRM conjugate (Lot A)        plus alum    -   Group 7—Type 5 capsular polysaccharide-CRM conjugate (Lot B)        plus alum

The conjugates of the invention (represented by lots A′, A and B) gaveprotection from kidney infection (FIGS. 13A and 13B). The conjugates ofthe invention gave high titres of specific IgG antibodies with lowtitres of IgM antibodies (FIG. 14).

Immunisation Study—Abscess Model (4)

CD1 mice at 3 weeks old were immunised at days 1 and 14 byintraperitoneal injection with a 1 μg dose of antigen in an injectionvolume of 200 μl. The mice were bled on days 0, 13 and 27 and challengedwith S. aureus on day 28. Organs were harvested at day 32. Immunisationswere carried out in groups of eight or nine mice according to thefollowing scheme:

-   -   Group 1—Type 8 capsular polysaccharide-CRM conjugate (lot α)        plus alum    -   Group 2—Type 8 capsular polysaccharide-CRM conjugate (lot α)        plus MF59    -   Group 3—Alum alone    -   Group 4—MF59 alone

The conjugates of the invention gave protection from kidney infection(FIG. 15). The alum formulation gave better protection than the MF59formulation.

Immunisation Study—Lethal Model (1)

General Assay Protocol:

Mice were immunized according to the schedule described below andchallenged by intraperitoneal injection of a bacterial suspension of S.aureus. Cultures of S. aureus were centrifuged, washed twice and dilutedin PBS before challenge. Further dilutions were needed for the desiredinoculum, which was experimentally verified by agar plating and colonyformation. Animals were monitored for 14 days and lethal diseaserecorded.

CD1 mice were immunised by intraperitoneal injection with a 5 μg dose ofantigen in an injection volume of 200 μl. Immunisations were carried outin groups of twelve mice according to the following scheme, prior tochallenge with 5×10⁸ CFU type 5 S. aureus:

-   -   Group 1—PBS plus alum    -   Group 2—Type 5 capsular polysaccharide-CRM conjugate (Lot C)        plus alum    -   Group 3—Type 5 capsular polysaccharide-CRMadh conjugate (Lot 3)        plus alum

The conjugates of the invention (represented by lot C) gave highersurvival (FIG. 16).

Immunisation Study—Lethal Model (2)

CD1 mice were immunised by intraperitoneal injection with a 2 μg(saccharide) and 10μ (protein, where present) doses of antigen in aninjection volume of 200 μl. Immunisations were carried out in groups oftwelve mice according to the following scheme, prior to challenge with5×10⁸ CFU type 5 S. aureus:

-   -   Group 1—PBS plus alum    -   Group 2—Type 5 capsular polysaccharide-CRM conjugate (Lot D)        plus alum    -   Group 3—Type 5 capsular polysaccharide-CRMadh conjugate (Lot 4)        plus alum    -   Group 4—Type 5 capsular polysaccharide-CRM conjugate (Lot D)        plus EsxAB, Sta006 and Sta011 proteins and alum    -   Group 5—Type 5 capsular polysaccharide-CRM conjugate (Lot D)        plus HlaH35L, Sta006 and Sta011 proteins and alum

Survival data is presented in Table 5.

TABLE 5 Time (days) Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 100 25 1717 17 17 17 17 17 17 8 0 0 0 2 100 50 50 50 50 50 50 50 50 42 42 42 4242 3 100 50 42 42 42 42 42 42 42 33 33 33 33 33 4 100 67 67 67 67 67 6767 67 67 67 67 67 67 5 100 100 100 100 100 100 83 83 75 75 75 75 75 75

The conjugates of the invention (represented by lot D) gave highersurvival. Survival was enhanced by addition of S. aureus proteinantigens.

Immunisation Study—Lethal Model (3)

CD1 mice were immunised by intraperitoneal injection with a 2 μg (type 5polysaccharide) 1 μg (type 8 polysaccharide, where present) and 10μ(protein, where present) doses of antigen in an injection volume of 200μl. Immunisations were carried out in groups of twelve mice according tothe following scheme, prior to challenge with 5×10⁸ CFU type 5 S.aureus:

-   -   Group 1—PBS plus alum    -   Group 2—Type 5 capsular polysaccharide-CRM conjugate (lot E)        plus EsxAB, Sta006 and Sta011 proteins and alum    -   Group 3—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot β) plus EsxAB,        Sta006 and Sta011 proteins and alum    -   Group 4—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot (3) plus        EsxAB, Sta011 and Sta073 proteins and alum    -   Survival data is presented in Table 7.

TABLE 7 Time (days) Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 100 50 4242 42 42 42 42 42 42 33 33 33 33 2 100 42 42 42 42 42 42 42 42 42 33 3333 33 3 100 75 75 75 75 75 75 75 75 75 58 50 50 50 4 100 92 92 83 83 8383 83 83 83 75 75 75 75

Immunisation Study—Lethal Model (4)

CD1 mice were immunised by intraperitoneal injection with a 2 μg (type 5capsular polysaccharide) 1 μg (type 8 capsular polysaccharide, wherepresent) and 10μ (protein, where present) doses of antigen in aninjection volume of 200 μl. Immunisations were carried out in groups oftwelve mice according to the following scheme, prior to challenge with5×10⁸ CFU type 5 S. aureus:

-   -   Group 1—PBS plus alum    -   Group 2—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot γ first dose,        lot δ second dose) plus EsxAB, Sta006 and Sta011 proteins and        alum    -   Group 3—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot γ first dose,        lot δ second dose) plus alum    -   Group 4—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot γ first dose,        lot δ second dose) plus EsxAB protein and alum    -   Group 5—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot γ first dose,        lot δ second dose) plus Sta006 protein and alum    -   Group 6—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot γ first dose,        lot δ second dose) plus Sta011 protein and alum    -   Group 7—Type 5 capsular polysaccharide-CRM conjugate (lot E) and        Type 8 capsular polysaccharide-CRM conjugate (lot γ first dose,        lot δ second dose) plus Sta006 and Sta011 proteins and alum    -   Group 8—Type 5 capsular polysaccharide-CRM conjugate (lot E)        plus HlaH35L, Sta006 and Sta011 proteins and alum    -   Group 9—Type 5 capsular polysaccharide-CRM conjugate (lot E)        plus HlaH35L protein and alum    -   Survival data is presented in Table 8.

TABLE 8 Time (days) Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 100 100 1313 13 13 13 13 13 13 13 13 13 13 2 100 88 75 75 63 63 63 50 50 50 50 5050 50 3 100 100 63 63 38 38 38 38 38 38 38 38 38 38 4 100 100 75 75 7575 75 75 63 50 50 25 25 25 5 100 100 50 50 50 50 50 38 38 38 38 38 38 386 100 100 25 25 25 25 25 25 25 25 25 13 13 13 7 100 88 63 63 63 63 63 5050 50 50 50 50 50 8 100 100 100 100 88 88 88 88 88 75 75 75 75 75 9 10088 88 63 38 38 38 38 38 13 13 13 13 13

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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The invention claimed is:
 1. A process of preparing a conjugate of anisolated fragment of the type 5 capsular polysaccharide ofStaphylococcus aureus and a carrier molecule comprising the steps of (a)depolymerising an isolated full-length type 5 capsular polysaccharide ofS. aureus to provide an isolated fragment of the type 5 capsularpolysaccharide having a molecular mass of between 5 kDa and 100 kDa; (b)oxidising the fragment of the type 5 capsular polysaccharide in order tointroduce an aldehyde group into at least one saccharide residue in thefragment to have at least one oxidised saccharide residue in thefragment; and (c) coupling the fragment having the at least one oxidisedsaccharide residue to the carrier molecule via the aldehyde group,thereby preparing the conjugate.
 2. A process of producing a purifiedfragment of a type 5 capsular polysaccharide of Staphylococcus aureuscomprising the step of depolymerizing a purified full-length type 5 S.aureus capsular polysaccharide by acid hydrolysis under conditionssuitable for the depolymerization, thereby producing the fragment of thepurified type 5 capsular polysaccharide of S. aureus, wherein thefragment has a □-D-FucNAc-(1→moiety at its non-reducing terminus.
 3. Theprocess of claim 2, wherein the acid hydrolysis comprises treating thefull-length purified type 5 S. aureus capsular polysaccharide with 2%(v/v) acetic acid at 90° C. for 3 hours or overnight.
 4. The process ofclaim 1, wherein the depolymerising is carried out by acid hydrolysisusing acetic acid.
 5. The process of claim 1, wherein the fragment has adegree of 0-acetylation of 10% to 90%.
 6. The process of claim 1,wherein the step (a) comprises depolymerising the full-length type 5 S.aureus capsular polysaccharide to provide the fragment of the type 5 S.aureus capsular polysaccharide having a □-D-FucNAc-(1→moiety at itsnon-reducing terminus.
 7. The process of claim 6, wherein the step (b)comprises oxidising the fragment of the type 5 S. aureus capsularpolysaccharide having the □-D-FucNAc-(1→moiety at its non-reducingterminus to convert the two vicinal hydroxyl groups in the□-D-FucNAc-(1→moiety into two aldehyde groups.
 8. A method of providingan oxidised Staphylococcus aureus type 5 capsular polysaccharidecomprising the step of oxidising an isolated S. aureus type 5 capsularpolysaccharide having a □-D-FucNAc-(1→moiety at its non-reducingterminus to convert the two vicinal hydroxyl groups in the□-D-FucNAc-(1→moiety into two aldehyde groups.
 9. A process of providinga coupled Staphylococcus aureus type 5 capsular polysaccharidecomprising the step of coupling to a carrier molecule an isolated S.aureus type 5 capsular polysaccharide having a □-D-FucNAc-(1→moiety atits non-reducing terminus that has been oxidised to convert its twovicinal hydroxyl groups into two aldehyde groups, wherein the couplingis via one of the aldehyde groups.
 10. The process of claim 1, whereinthe coupling is direct coupling by reacting the aldehyde group with anamine group in the carrier molecule by reductive amination.
 11. Theprocess of claim 1, wherein the coupling is via a linker by reacting thealdehyde group with an amine group in the linker by reductive amination.12. The process of claim 11, wherein the linker is already attached tothe carrier molecule.
 13. The process of claim 1, wherein the carriermolecule is a carrier protein and wherein the coupling results in aratio of the fragment of the type 5 S. aureus capsular polysaccharide tothe carrier protein (w/w) of between 1:5 and 1:2.
 14. The process ofclaim 11, wherein the linker attached to the fragment of the capsularpolysaccharide is then attached to the carrier molecule.